ES2254176T3 - METHODS FOR MAKING COMPOSITIONS WITH A CONDITIONING CELL CULTURE. - Google Patents

Abstract

A method of preparing a pharmaceutical composition comprising: (a) culturing three-dimensional fibroblast cells to form a three-dimensional stroma tissue in a cell culture medium sufficient to meet the nutritional needs required for in vitro cell growth and wherein the stromal cells are attached to a structure composed of a non-living biocompatible material formed in a three-dimensional structure and subsequently surround said structure; (b) culturing the cells in vitro until the cell culture medium contains a desired level of extracellular products so that a conditioned medium is formed; (c) separating the conditioned medium from the cells used to condition the medium; and (d) combine the conditioned medium with a pharmaceutical support.

Description

Methods for making compositions using conditioned cell culture.

1. Introduction

The invention relates to methods for making compositions comprising a cell culture medium conditioned by the growth of cells in a culture in two dimensions (i.e. a monolayer), or in a crop in three dimensions. The cells used to condition the medium can be genetically modified to alter concentration of proteins found in the medium. The conditioned cellular medium is processed for uses that include wound applications, cosmetic additives, food supplements, supplements for animal feed, crop cells, applications pharmaceuticals, as well as compositions and methods to stimulate hair growth

2. Background of the invention 2.1. Conditioned Cellular Medium

Compositions of culture media typically include essential amino acids, salts, vitamins, minerals, trace metals, sugars, lipids and nucleosides. Cell culture media attempt to provide the necessary components to meet the nutritional needs required for cell growth in a controlled, artificial and in vitro environment . Nutrient formulations, pH values, and osmolarity vary according to parameters such as cell type, cell density, and the culture system employed. Many formulations of culture media are documented in the literature and a good number of media are commercially available. Once the culture medium is incubated with cells, it is known to those skilled in the art as "spent" or "conditioned" media. The conditioned media contain many of the original components of the medium, as well as a variety of cellular metabolites and secreted proteins, including, for example, biologically active growth factors, inflammatory mediators and other extracellular proteins. Cell lines grow as a monolayer or in "pearls", as opposed to cells that grow in three dimensions, which lack the cell-cell and cell-matrix interactions characteristic of whole tissue in vivo . Consequently, said cells secrete a variety of cellular metabolites although they do not necessarily secrete these metabolites and the secreted proteins at levels that approach physiological levels. The conditioned cell culture media, the culture media by cell line growth as a monolayer or in "beads", are normally discarded or used occasionally in crop manipulations such as cell density reduction.

2.2. Tissue Cultivation Systems

Most in vitro vertebrate cell cultures are grown as monolayers in an artificial substrate bathed in the culture medium. The nature of the substrate on which the monolayers grow can be solid, such as plastic, or semi-solid gels, such as collagen or agar. Disposable plastics have been transformed into the currently preferred substrate used in tissue or cell cultures.

A few researchers have explored the use of natural substrates related to basement membrane components. The base membranes comprise a mixture of glycoproteins and proteoglycans that surround most of the cells in vivo . For example, Reid and Rojkund, 1979, in, Methods in Enzymology , Vol. 57, Cell Culture , Jakoby & Pasten, eds., New York, Acad. Press, p. 263-278; Vlodavsky et al., 1980 Cell 19: 607-617; Yang et al., 1979, Proc. Natl Acad. Sci. USA 76: 3401 have used collagen for the cultivation of hepatocytes, epithelial cells and endothelial tissue. Floating collagen cell growth (Michalopoulos and Pitot, 1975, Fed. Proc. 34: 826) and cellulose nitrate membranes (Savage and Bonney, 1978, Exp. Cell Res . 114: 307-315) has been used in attempts to favor terminal differentiation. However, until now, prolonged cell regeneration and culture of said tissues in said systems has not been achieved.

Cultures of mouse embryo fibroblasts have been used to increase cell growth, particularly at low densities. It is thought that this effect is partly due to the supplementation of the medium but it may also be due to the conditioning of the substrate by cellular products. In these systems, the fibroblast feeder layers are grown as confluent monolayers which makes the surface suitable for the binding of other cells. For example, glioma growth in confluent feeder layers of normal fetal intestine has been described (Lindsay, 1979, Nature 228: 80).

Although the growth of cells in two dimensions is a convenient method for the preparation, observation and study of cells in culture, allowing a high percentage of cell proliferation, it lacks the characteristics of the whole tissue in vivo . In order to study such functional and morphological interactions, some researchers have explored the use of three-dimensional substrates such as collagen gel (Douglas et al., 1980, In Vitro 16: 306-312; Yang et al., 1979, Proc. Natl. Acad. Sci. 76: 3401; Yang et al., 1980, Proc. Natl. Acad. Sci. 77: 2088-2092; Yang et al., 1981, Cancer Res. 41: 1021-1027 ); cellulose sponge, alone (Leighton et al., 1951, J. Natl. Cancer Inst. 12: 545-561) or coated collagen (Leighton et al., 1968, Cancer Res . 28: 286-296); a jelly sponge, Gelfoam (Sorour et al., 1975, J. Neurosurg. 43: 742-749).

In general, these three-dimensional substrates are inoculated with the cells to be cultured. It has been described that many of the cell types penetrate the matrix and establish a "tissue type" histology. For example, three-dimensional collagen gels have been used to cultivate chest epithelium (Yang et al., 1981, Cancer Res. 41: 1021-1027) and sympathetic neurons (Ebendal, 1976, Exp. Cell Res . 98: 159 -169). Additionally, several attempts have been made to regenerate the tissue-like architecture from dispersed monolayer cultures. (Kruse and Miedema, 1965, J. Cell. Biol . 27: 273) have described that the pre-fused monolayers can grow to a depth of more than ten cells and the organoid structures can develop in multilayer cultures if they are kept fed with a medium appropriate (see also Scheneider et al., 1963, Exp. Cell. Res. 30: 449-459; Bell et al., 1979, Proc. Natl. Acad. Sci. USA 76: 1274-1279; Green, 1978, Science 200: 1385-1388). It has been described that human epidermis keratonocytes can form dematoglyphs (friction ridges if held for several weeks without transfer; Folkman and Haudenschild (1980, Nature 288: 551-556) have described the formation of capillary tubules in endothelial cell cultures vascular cultures grown in the presence of endothelial growth factor and conditioned medium for tumor cells; and Sirica et al., (1979, Proc. Natl. Acad. Sci . USA 76: 283-287; 1980, Cancer Res . 40: 3259 -3267) maintained hepatocytes in a primary culture for approximately 10-13 days in nylon meshes coated with a thin layer of collagen, however, long-term culture and cell proliferation in such systems has not been achieved.

The establishment of long-term culture of tissues such as bone marrow has been attempted. The overall results were discouraging, since although the stromal cell layer that contains different types of cells forms rapidly, a significant hematopoiesis cannot be maintained for any real time. (For review see Dexter et al., In Long Term Bone Marrow Culture , 1984, R. Alan, Liss, Inc., pp. 57-96).

A number of groups have tried to grow skin and connective tissue in vitro for transplants in vivo . In one of such systems, a hydrated bovine collagen framework forms the substrate into which cells, such as fibroblasts, are incorporated which results in tissue contraction in the tissue (Bell et al., US Patent No. 4,485,096). In another system, a porous crosslinked collagen sponge is used to grow fibroblast cells (Eisenberg, WO 91/16010). A scaffolding composed of synthetic polymers to control cell growth and proliferation in vitro has also been described so that once the fibroblasts begin to grow and attack the matrix it is transplanted to the patient (Vacanti et al., US Patent No. 5,759,830; 5,770,193; 5,736,372).

Synthetic matrices composed of biodegradable, biocompatible copolymers of polyesters and amino acids have also been designed as scaffolding for cell growth (US Patent Nos. 5,654,381; 5,709,854). Non-biodegradable scaffolds are equally capable of supporting cell growth. Three-dimensional cell culture systems composed of a stroma matrix that support the growth of cells from the desired tissue in an adult tissue have also been designed (Naughton et al., US Pat. Nos. 4,721,096 and 5,032,508 ). Another approach involves the slow polymerization of hydrogels containing a large number of desired cell types that harden in a matrix once administered to a patient (US Patent No. 5,709,854). Extracellular matrix preparations have been designed so that they are composed of stroma cells that provide a three-dimensional cell culture system for a desired cell type that can be injected into the patient to precisely position the biomaterial (Naughton et al. , WO 96/39101).

2.3. Cellular Cytokines and Growth Factors

The secretion of extracellular proteins in conditioned cell media such as growth factors, cytokines, and stress proteins opens up new possibilities in the preparation of products for use in a wide variety of areas including tissue repair, e.g. , in the treatment of wounds and other tissue defects such as cosmetic defects as well as human and animal nutritional supplements. For example, it is known that growth factors play an important role in the wound healing procedure. In general, it is thought that it is desirable in wound treatment to intensify the growth factor supplement by direct addition of these factors.

Cellular cytokines and growth factors are involved in a number of critical cellular procedures including cell proliferation, adhesion, morphological appearance, differentiation, migration, inflammatory responses, angiogenesis, and cell death. Studies have shown that hypoxic tension and cell damage induces responses that include increased levels of mRNA and proteins corresponding to growth factors such as PDGF (platelet-derived growth factor), VEGF (vascular endothelial growth factor ), FGF (fibroblast growth factor), and IGF (insulin type growth factor) (M. González-Rubio, et al. , 1996, Kidney Int . 50 (1): 164-73; R. Abramovitch, et al. , 1997, Int J. Exp. Pthol. 78 (2): 57-70; I. Stein et al. , 1995; Mol Cell Biol. 15 (10): 5363-8; W. Yang et al. , 1997, FEBS Lett. 403 (2): 139-42; NR West et al., 1995, J. Neurosci. Res. 40 (5): 647-59).

Growth factors, such as transforming growth factor?, Also known in the state of the art as TGF-?, Are induced by certain tension proteins during wound healing. Two known stress proteins are GRP78 and HSP90. These proteins stabilize cell structures and give rise to cells resistant to adverse conditions. The TGF-? Family of dimeric proteins includes TGF-? 1, TGF-? 2, and TGF-? 3 and regulates the growth and differentiation of many cell types. In addition, this family of proteins shows a range of biological effects, stimulating the growth of some cell types (Noda et al. , 1989, Endocrinology 124 : 2991-2995) and inhibiting the growth of other cell types (Goey et al. , 1989, J. Immunol. 143 : 877-880; Pietenpol et al. , 1990, Proc. Natl. Acad. Sci. USA 87 : 3758-3762). TGF-? Has also been shown to increase the expression of extracellular matrix proteins including collagen and fibronectin (Ignotz et al. , 1986, J. Biol. Chem. 261 : 4337-4345) and to accelerate healing of wounds (Mustoe et al., 1987, Science 237 : 1333-1335).

Another such growth factor is the PDGF. Originally, PDGF was found to be a potent mitogen for mesenchyme derived cells (R. Ross et al ., 1974, Proc. Natl. Acad. Sci. USA 71 (4): 1207-1210; N. Kohler et al . , 1974, Exp. Cell Res. 87 : 297-301). Additional studies have shown that PDGF increases the ratio of speed and granulation in tissue formation. Wounds treated with PDGF have the appearance of a primary-state inflammatory response including an increase in types of neutrophil cells and macrophages at the wound site. These wounds have also shown an enhanced fibroblast function (GF Pierce, et al ., 1988, J. Exp. Med. 167 : 974-987). Both PDGF and TGF-? Have been shown to increase collagen formation, DNA content, and protein levels in animal studies (GR Grotendorst et al ., 1985, J. Clin. Invest. 76 : 2323- 2329; MB Sport et al. , 1983, Science (Wash DC) 219 : 1329). The PDGF has proven effective in treating human wounds. In human wounds, the expression of PDGF-AA is increased with the pressure of the ulcers that are healed. The increase in PDGF-AA corresponds to an increase in activated fibroblasts, the deposition of the extracellular matrix, and the active vascularization of the wound. In addition, such an increase in PDGF-AA is not observed in chronic unhealed wounds (Principles of Tissue Engineering, R. Lanza et al . (Eds), pp. 133-141 (RG Landes Co. TX 1997). Other growth factors that have the ability to induce angiogenesis and wound healing include VEGF, KGF and basic FGF.

There are currently no simple effective methods or compositions for the application containing the variety of cytokines, growth factors or other regulatory proteins in the conditioned media of the Applicants.

3. Summary of the invention

Applicants of the present invention have discovered new methods for the production of compositions of conditioned cell culture media.

The methods for making the conditioned cell media compositions of the invention may be comprised of any known defined or undefined media and may be conditioned using any type of eukaryotic cell. The medium may be conditioned by stromal cells, parenchymal cells, mesenchymal germ cells, liver reserve cells, neural germ cells, pancreatic germ cells, and / or non-human embryonic germ cells. A three-dimensional fabric construction was used. The type of cell will affect the properties of the conditioned medium. For example, a medium conditioned with astrocytes and neuronal cells will make certain characteristic metabolites and proteins so that said conditioned medium is preferred for certain nerve repair applications. The applicants' medium is conditioned with a culture of cells and tissues in three dimensions. In another preferred embodiment, the medium is conditioned with the stromal cells used in the production of TransCyte® (Smith & Nephew PLC., United Kingdom). In a highly preferred embodiment, the three-dimensional tissue culture cells are stroma cells and the tissue culture construct culture is Dermagraft® (Advanced Tissue Sciences, Inc. La Jolla CA) with or without the addition of cells. of specific parenchyma. Such conditioned cell media provide a combination of specified factors and ratios that are different from monolayer cultures and represent closer to those found in vivo . Three-dimensional stromal cultures can be further cultured with parenchymal cells such as skin, bone, liver, nerve, pancreas, etc. cells, resulting in a conditioned medium containing extracellular proteins. characteristics and other metabolites of said type of tissue. Additionally, each type of cell can also be genetically modified. The genetic modification can be used to alter the concentration of one or more components in the medium such as, for example, to over-regulate a protein, to introduce a new protein, or to regulate the ion concentration.

Once the cellular medium, obtained according to The invention, is conditioned, can be used in any state. Physical embodiments of the conditioned medium include, but are not limited to, liquid or solid, frozen, lyophilized, or dried in a powder. Additionally, the medium can be formulated with a pharmaceutically acceptable carrier as a vehicle for internal administration, applied directly to a food item or product, formulated with an ointment or ointment for topical applications, or, for example, prepared in or added to a surgical glue to accelerate the healing of Sutures after invasive procedures. Also, the middle can be processed additionally to concentrate or reduce one or more factors or components contained in the medium. For example, the conditioned medium can be enriched with a factor of growth by using chromatography of immunoaffinity

In one embodiment, the conditioned medium obtained by the invention is used in the healing of wounds Examples include, but are not limited to, the application of the conditioned cellular media to the gauze of a bandage (adhesive or non-adhesive) and used in topical applications for favor and / or accelerate wound healing. Again the middle conditioning can be processed to concentrate or reduce one or more components to intensify wound healing. The compositions can be lyophilized and added as a filler of wounds or added to wound filling compositions Existing to accelerate wound healing. So alternatively the medium can be added to a hydrogel composition and used as a film for topical treatments of wounds and for anti-adhesion applications. The media compositions of the invention can be conditioned with cells expressing gene products with properties of improved wound healing; that is, engineering cells genetics that express gene products that have properties anti-scarring

In another embodiment, the media formulations conditioned cells obtained by the invention are used to correct congenital abnormalities and physical defects acquired. In addition, formulations in the form of injectables or hydrogels to eliminate wrinkles, lines of expression, scars and to repair other skin conditions. In another embodiment the conditioned cellular medium can also be added to an eye shadow, a cream or makeup powder or other cosmetics

In yet another embodiment, the formulations of the conditioned cellular media obtained by the invention are used as food additives and dietary supplements. He conditioned medium contains a multitude of nutrients including essential amino acids, vitamins and minerals. Cellular media conditioners of the invention can be concentrated and / or lyophilized, for example, and are preferably administered in capsules or tablets for ingestion. Additionally the compositions can also be added directly to food to intensify its nutritional content.

In a further embodiment, the compositions can be used as a supplement for animal foods since they contain a variety of proteins, vitamins, antibiotics, polysaccharides and other beneficial factors for livestock and other ruminant animals

In yet another embodiment of the invention, the compositions obtained by the invention can be used for Cell cultures. The conditioned cellular media of the invention contain useful factors in favoring the binding of cells and growth. In addition, the cellular medium can be conditioning by cells that are the result of genetic engineering and which may contain, for example, fibronective concentrations,  or collagen, increased, beneficial in promoting the union of cells to a scaffold or culture surface.

In a further embodiment of the invention, the compositions of the conditioned cellular medium obtained by the invention are used for pharmaceutical applications. The invention comprises cell media cultured with three-dimensional tissue constructs, so that growth factors and other proteins are secreted in the medium at speeds closely similar to those found in vivo. Thus, the conditioned media of the invention are beneficial for a variety of pharmaceutical applications.

Finally, the compositions obtained by the invention can be formulated for topical applications for hair growth stimulation.

3.1. Definitions

The following terms used herein invention have the meanings indicated:

Adherent Layer : cells directly attached to the three-dimensional support or indirectly connected by binding to cells that are directly attached to the support themselves.

Conditioned Medium : a formulation containing extracellular protein (s) and cellular metabolites, which has previously supported the growth of any desired eukaryotic cell type, so that said cells have been cultured in both two and three dimensions. Also called "Conditioned Cellular Medium" or "Medium of Cells or of Conditioned Tissue Culture".

Stroma cells : fibroblasts with or without other cells and / or elements found in loosened connective tissues, including, but not limited to, endothelial cells, pericytes, macrophages, monoliths, plasma cells, mast cells, adiposites, mesemchyme germ cells, cells reserve liver, neuronal germ cells, pancreatic germ cells, condorites, prechondrocytes, etc.

Parenchyma or Tissue-specific Cells: the cells that form the essential and distinctive tissue of an organ as distinguished from its support structure.

Tri-Dimensional Structure : a three-dimensional scaffolding composed of any material and / or shape that (a) allows cells to bind to it (or that can be modified to allow cells to bind to it); and (b) allow cells to grow in more than one layer. This support is inoculated with stroma cells to form live stromal tissue of three dimensions. The framework structure may include a mesh, a sponge or it may be formed from a hydrogel.

Three-dimensional Stroma Tissue or Live Stroma Matrix : a three-dimensional structure that has been inoculated with stroma cells that have been grown in the support. Extracellular matrix proteins made by stroma cells are deposited in the structure, thereby forming a live stroma tissue. Live stromal tissue can support the growth of tissue specific cells inoculated later to form the culture of three-dimensional cells.

Tissue Culture in Three-Dimensional Tissue Specific or Tri-Dimensional Tissue Specific Construction : a three-dimensional live stroma tissue that has been inoculated with tissue-specific and cultured cells. In general, tissue-specific cells used to inoculate the three-dimensional stroma matrix should include "germ cells" (or "reserve" cells) for said tissue; that is, those cells that generate new cells that will mature in the specialized cells that form the tissue parenchyma.

Those root cells are not obtained from of human embryos.

The following abbreviations must have the indicated meanings:

BCS = bovine serum

BFU-E = burst - unit trainer - erythroid

TGF-? = Factor transforming growth?

CFU-C = forming unit colonies - cultivation

CFU-GEMM = forming unit of colonies - granuloid, erythroid, monolith, megakaryocyte

CSF = colony stimulating factor

DMEM = Dulbecco's Modified Eagle Medium

EDTA = ethylene diamine tetraacetic acid

FBS = fetal bovine serum

FGF = fibroblast growth factor

GAG = glycosaminoglycan

GM-CSF = stimulating factor of granulocyte / macrophage colonies

HBSS = Hank's balanced salt solution

HS = horse serum

IGF = insulin-like growth factor

LTBMC = long bone marrow culture finished

MEM = minimum essential medium

PBL = peripheral blood leukocytes

PBS = phosphate buffered saline

PDGF = growth factor derived from platelets

RPMI 1640 = middle of the Roswell Park Institute Memorial number 1640 (GIBCO, Inc., Grand island, N.Y.)

SEM = scanning electron microscopy

VEGF = endothelial growth factor vascular

4. Description of the figures

Fig. 1 is a graph that represents the deposition kinetics of glycosaminoglycans and collagen released over time by the woven products of three Transcyte® and Dermagraft® dimensions. The deposition volume of glycosaminoglycans is dependent on the period of growth while collagen deposition is not dependent on growing period.

Fig. 2 is a graph that represents the effect of the extracellular matrix (separate from Transcyte®) and added to dilutions of 1: 2, 1: 5, 1:10, and 1: 100 to monolayer cultures of Human fibroblasts and keratinocytes. The most significant effect Illustrated is at a 1:10 dilution of the matrix.

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Fig. 3 is a graph representing the relative proliferation of human fibroblasts and keratinocytes exposed to the conditioned medium (cell culture medium that has previously supported cell growth in Transcyte®). Be revealed an increase in cell response in such a time Short as three days.

Fig. 4 is a graph that represents the effect of the 1 X conditioned medium (cell culture medium that He has previously supported the growth of cells in Transcyte®) in Collagen deposition of cell growth in three dimensions compared to the DMEM base medium (containing a 10% BLS supplemented medium with 2 mM L-glutamine and antibiotic / antifungal 1 X) supplemented with a concentration 1 X final of medium and serum free medium. An increase was noticed statistically significant (p = 0.05) of almost 50% in the collagen deposition of cultures treated with the medium conditioning for 10 days compared to the control.

Fig. 5 is a graph representing the anti-oxidant activity in a cell medium conditioning (cell culture medium that has supported previously the growth of cells in Transcyte®) in Human epidermal keratinocytes in culture. It noticed a statistically significant reduction (p <0.0003) in the intracellular oxidation of approximately 50% in keratinocytes exposed to the conditioned medium that has been previously supported in Transcyte® for three days.

5. Detailed description of the invention

The present invention relates to methods for the preparation of compositions comprising any medium conditioning defined or undefined, grown using any eukaryotic cell type of a construction of a tissue of three dimensions and methods for the use of the compositions. The cells are cultured in three dimensions. The cells are preferably human to reduce the risk of a response immune and include stroma cells, parenchyma cells, mesenchymal germ cells, liver reserve cells, neural germ cells, pancreatic germ cells and / or non-human embionic germ cells. The medium conditioned by the cell and tissue cultures will contain a variety of naturally secreted proteins, such as factors of active growth and those grown in three dimensions that they will have these proteins in close relationships to levels physiological The invention also relates to new methods for prepare compositions comprising media derived products of conditioned cells and uses for these compositions.

The "pre-conditioned" cell culture media can be any cell culture medium that adequately addresses the nutritional needs of the cells to be cultured. Examples of cell media include, but are not limited to, Dulbecco Modified Tagle Medium (DMEM), Ham F12, RPMI 1640, Iscove, McCoy and other readily apparent media formulations for those skilled in the state of technique, including those found in Methods For Preparation of Media, Supplements and Substrate For FERUM-Free Animal Cell Culture R. Liss, New Cork (1984) and Cell & Tissue Culture: Laboratory Procedures , John Wiley Sons Ltd., Chichester, England 1996 The medium can be supplemented, with any of the components necessary to support the desired cell or tissue culture. Additionally, serum, such as bovine serum, which is a complex solution of albumins, globulins, growth promoters and growth inhibitors may be added if desired. Serum should be pathogen-free and carefully selected to prevent contamination of mycoplasma bacteria, fungi and viruses. Also, the serum must be obtained in general from the United States and must not be obtained from countries where the way of life of the indigenous people can transmit agents. The addition of hormone in the medium may or may not be desirable.

Other ingredients can be selected, such like vitamins, growth and binding factors, proteins, etc., by experts in the field according to their particular need. The present invention can use any type appropriate cell to achieve the desired conditioned medium. Genetic engineering cells can be used to grow the media. Said cells can be modified, for example, to express a desired protein or proteins so that the concentration of the protein or proteins expressed in the medium be optimized for the particular application you want. Agree with the present invention, cell and tissue cultures used to condition the medium can be modified by genetic engineering to express a target gene product that can impart a wide variety of functions, including, but not limited to, improved protein expression properties that have similar physiological reactions, the expression increased of a particular protein useful for an application specific, such as wound healing or inhibition of certain proteins, such as proteases, lactic acid, etc.

Cells can be engineered genetics to express a target gene product that is biologically active and that provides a biological function selected, acting as a reporter of a physiological state selected, which increases the defective or defective expression of a product of a gene, or that provides an activity anti-viral, anti-bacterial, or anti-cancer In accordance with the present invention, the product of the target gene can be a peptide or a protein, such as a transcription factor or a DNA binding protein, a structural protein, such as a surface protein cell, or the product of the target gene can be a nucleic acid such as a ribosome or an antisense molecule. The products of target gene include, but are not limited to, gene products that intensify cell growth. For example, the genetic modification can over-regulate a endogenous protein, introduce a new protein or regulate the ion concentration by expression of a heterologous ion channel or by altering the function of the endogenous ion channel. Examples include, but are not limited to fabrics modified by genetic engineering that express the gene products that are systematically released (e.g., gene products segregated such as proteins including growth factors, hormones, Factor VIII, Factor IX, neurotransmitters, and encafalinas).

In the present invention, it is preferred that the cells grow on a three-dimensional stroma support and grow on multiple layers, forming a cellular matrix. This matrix system approximates the physiological conditions found in vivo to a greater degree than the previously described monolayer tissue culture systems. Three-dimensional cultures, such as Dermagraft® (Advanced Tissue Sciences, Inc., La Jolla, CA) "Dermagraft®", and TransCyte® (Smith & Nephew, PLC, UK) "TransCyte®", produces numerous factors of growth and other proteins that are secreted in the medium at physiological ratios and concentrations. Dermagraft® is composed of allogeneic neonatal fibroblasts cultured in biodegradable polyglactin. TransCyte® is a temporary substitute for living skin that comprises a three-dimensional stroma tissue attached to a transitional cover as described in US Patent 5,460,939. Additionally, three-dimensional tissue cultures that condition cell media may contain mesenchymal germ cells, liver reserve cells, neural germ cells, pancreatic germ cells, and / or embryonic germ cells and / or parenchyma cells and / or parenchymal germ cells found in many types of tissues, including but not limited to, bone marrow, skin, liver, pancreas, kidney, adrenal and neurological tissue, as well as tissues of the gastrointestinal and genitourinary tracts, and the circulatory system . See United States Patents Nos. 4,721,096; 4,963,489; 5,032,508; 5,266,480; 5,160,490 and 5,559,022.

5.1. Cellular Media

The formulations of the culture media cell phones are well known in the literature and many are commercially affordable

Pre-conditioned media include, but are not limited to, those described below. Additionally, the concentrations of the ingredients are well known to a person skilled in the art. See, for example, Methods For Preparation Of Media, Supplements and Substrate for FERUM-free Animal Cell Cultures, supra . The ingredients include amino acids (both amino acids D and / or L-) such as glutamine, alanine, arginine, asparagine, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine , threonine, tryptophan, tyrosine, and valine and its derivatives; acid-soluble subgroups such as thiamine, ascorbic acid, ferric compounds, ferrous compounds, purines, glutathione and monobasic sodium phosphates.

Additional ingredients include sugars, deoxyribose, ribose, nucleosides, water soluble vitamins, riboflavin, traces of metals, lipids, acetate salts, salts of phosphate, HEPES, phenol red, pyruvate salts and buffers.

Other ingredients often used in Media formulations include fat soluble vitamins (including A, D, E and K) steroids and their derivatives, cholesterol, fatty acids and lipids Tween 80, pyramids of 2-mercaptoethanol as well as a variety of supplements including serum (fetal, horse, calf, etc.), proteins (insulin, transferred, growth factors, hormones, etc.), antibiotics (gentamicin, penicillin, streptomycin, amphotericin B, etc.), ultra-filtered whole egg, and factors binding (fibronectins, vitronectins, collagens, laminins, tenascinas, etc.).

Naturally, the media can specify, or not, be supplemented with growth factors and other proteins such as binding factors since many of the constructions of cells, particularly cell culture constructs and three-dimensional fabrics described in this application elaborate by themselves said growth and binding factors and others media products as discussed in greater detail to then Section 5.8.

5.2. Cell Cultures 5.2.1. The cells

The media may be conditioned by stromal cells, parenchymal cells, mesenchymal germ cells (assigned or unassigned lineage progenitor cells), liver reserve cells, neural germ cells, pancreatic germ cells, and / or embryonic germ cells not human Cells may include, but are not limited to, bone marrow, skin, liver, pancreas, kidney, neurological tissue, adrenal gland, mucosal epithelium, and smooth muscle to name a few. Fibroblasts and fibroblast-like cells and other cells and / or elements comprising the stroma may have a fetal or adult origin, and may be derived from convenient sources such as the skin, liver, pancreas, mucosa, arteries , veins, umbilical cord, and placental tissues, etc. Such tissues and / or organs can be obtained with an appropriate biopsy or by autopsy. In fact
Corpse organs can be used to provide a generous source of stromal cells and elements.

Non-human embryonic germ cells and / or other elements comprising the stroma can be isolated using methods known in the state of the art. Isolation and culture of mesenchymal germ cells are known in the state of the art. See Mackay et al., Tissue Eng 4: 415-428 (1988); William et al., Am Surg. 65: 22-26 (1999). The inoculation of these cells is described below in Section 5.3. Similarly, neural germ cells can be isolated in the manner described in Flax et al., Nature Biotechnol ., 16: 1033-1039 (1998); and Frisen et al., Cell. Mol. Life Sci., 54: 935-945 (1998).

The cells can be cultured in any of the ways known in the state of the art including in monolayers, drops or in three dimensions and by any means (i.e. culture plates, bottles, a continuous flow system, etc.). Cell and tissue culture methods are well known in the state of the art, and are described, for example, in Cell & Tissue Culture: Laboratory Procedures , cited above; Freeshny (1987), Culture of Animal Cells: A Manual of Basic Technics , see below.

In general, the cell lines used are carefully selected for human and animal pathogens. Depending on the application, this selection can be critical importance in cases where only cells are acceptable free of pathogens (for example, for wound healing, for food additives, etc.). The selection methods of Pathogens are well known in the state of the art. The kind of cells, whether it is cultured in two dimensions or in three dimensions, will affect the properties of the conditioned medium. Be He prefers a three-dimensional construction.

5.2.2. Three-dimensional cell cultures

Stroma cells used in cultures in three dimensions comprise fibroblasts, germ cells of mesenchyme, liver reserve cells, germ cells neural, pancreatic germ cells, and / or germ cells embryonic, with or without additional cells and / or elements described in more detail below.

Fibroblasts will support the growth of many different cells and tissues in the culture system in three dimensions, and therefore can be inoculated in the matrix to form a "generic" stromal support matrix for the culture of any variety of cells and tissues. Without However, in certain cases, it is preferable to use a matrix of "specific" stroma support rather than "generic", in in which case the stromal cells and elements can be obtained from an organ, a particular tissue, or a individual. In addition, fibroblasts and other cells and / or elements of stroma can be derived from the same type of tissue to be grown in The three dimensional system. This can be advantageous when they grow tissues in which specialized stromal cells they can play particular structural / functional roles; by example, smooth muscle cells of arteries, glial cells of neurological tissue, and liver Kupffer cells, etc.

Once inoculated in the support in three dimensions, stromal cells will proliferate in the structure and will deposit connective tissue proteins of natural origin secreted by stromal cells such as factors of growth, regulatory factors and matrix proteins extracellular Stroma cells and their tissue proteins connectively segregated naturally envelop substantially the structure thus forming the live stroma tissue that will support the growth of tissue specific cells inoculated into the three-dimensional culture system of the invention. In fact, when inoculated with specific cells of the tissue, the three-dimensional stroma tissue will maintain the active proliferation of the crop for long periods of time. Importantly, because the openings in the mesh allow the exit of stroma cells in the culture, the confluent stromal cultures do not show an inhibition of contact and stromal cells continue to grow, divide, and remain functionally active.

Growth factors and regulators are made by stroma tissues in the media. The facts growth (for example, but not limited to,? FGF, βFGF, insulin growth factor or TGF-betas), or natural blood products or modified or other biologically active molecules (for example, but not limited to, hyaluronic acid or hormones), intensify the colonization of the structure or scaffolding in three dimensions and the condition of the culture media.

The extent to which stromal cells are grown before the use of in vivo cultures may vary depending on the type of tissue to be grown in the three-dimensional tissue culture. The live stroma tissues that condition the medium can be used as corrective structures by implanting them in vivo. Alternatively, live stromal tissues can be inoculated with another type of cell and implanted in vivo . Stroma cells can be engineered to adjust the level of segregated protein products in the culture medium to improve the concentration of recovered product obtained from the conditioned medium. For example, anti-inflammatory factors, for example, anti-GM-CSF, anti-TNF, anti-IL-1, anti-IL-2, etc. Alternatively, stromal cells can be engineered to eliminate the expression of native gene products that favor inflammation, for example, GM-CSF, TNF, IL-1, IL-2, or to eliminate MHC expression in order to decrease the risk of rejection.

Stromal cell growth in three dimensions will support active proliferation of both stromal cells and tissue-specific cells in cultures for much longer periods of time than monolayer systems. In addition, the three-dimensional system supports maturation, differentiation, and segregation of cells in in vitro cultures to form components of adult tissues analogous to counterparts found in vivo and ensure that the proteins in the conditioned medium have much more physiological relationships. it seemed
you give.

Although applicants are not required to explain the mechanism by which cells and tissues function in three dimensions, a number of factors inherent in the system of Three-dimensional cultivation can contribute to your success:

(a) The three-dimensional structure provides a larger surface area for protein binding, and consequently, for the adhesion of stroma cells; Y

(b) Due to the three-dimensionality of the structure, stromal cells continue to grow so activates, in contrast to monolayer culture cells, which they grow to converge, exhibit contact inhibition, and stop Grow and divide. The development of growth factors and regulators by stromal cell replication can be partially responsible for stimulating proliferation and regulating the differentiation of cells in culture;

(c) The three-dimensional structure allows a spatial distribution of cellular elements that is more analogous to that found in the counterpart tissue in vivo ;

(d) The increase in potential volume for the cell growth in the three-dimensional system can allow the establishment of conductive microenvironments to the cell maturation;

(e) The three-dimensional structure maximizes cell-cell interactions allowing greater potential for movement of migratory cells, such as macrophages, monoliths and possible lymphocytes in the layer adherent;

(f) It has been recognized that the maintenance of a Differentiated cell phenotype requires not only factors of growth / differentiation but also appropriate cellular interactions The present invention effectively recreates the tissue microenvironment that results in a conditioned medium higher.

5.3. Establishment of Stroma tissue in Three Dimensions

The support or structure in three dimensions it can be of any material and / or shape, so that: (a) it allows that the cells bind to it (or it can be modified so that allow cells to bind to it); and (b) allows the cells grow in more than one layer. A number of different materials to form the structure, including, but not limited to: non-biodegradable materials, e.g. nylon (polyamides), dracon (polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., chloride polyvinyl), polycarbonate (PVC), polytetrafluoroethylene (PTFE; Teflon), termanox (TPX), nitrocellulose, cotton; and materials biodegradable, e.g., polyglycolic acid (PGA), collagen, collagen sponges, cat gut sutures, cellulose, jelly, dextran, polyalkanoates, etc. Any of these materials can be knitted, knitted, etc. in a mesh, for example, to form The structure in three dimensions. The structure, in turn can be created in any desired way such as structure corrective, e.g. tubes, ropes, filaments, etc. Some Materials, such as nylon, polystyrene, etc., are substrates poor for cell union. When these materials are used As three-dimensional structures, it is recommended pre-treat the structure before inoculation of stromal cells in order to intensify the binding of stroma cells to the support. For example, before inoculation With stroma cells, nylon structures can be treated with 0.1 M acetic acid, and incubated in polylysine, FBS, and / or collagen to coat the nylon. Polystyrene can be treated similarly using sulfuric acid.

When cultures that condition the medium are to be implanted in vivo , it may be preferable to use biodegradable matrices such as, for example, polyglycolic acid, collagen, collagen sponges, woven collagen, cat gut suture material, gelatin, polylactic acid , or polyglycolic acid and copolymers thereof. When crops are to be maintained for long periods of time or cryopreserved, non-degradable materials such as nylon, dracon, polystyrene, polyacrylates, polyvinyl, Teflon, cotton, etc. may be preferred. A convenient nylon mesh that can be used in accordance with the invention is Nitex, a nylon filtration mesh having an average pore size of 210 µm and an average nylon fiber diameter of 90 µm (# 3 -210/36, Tetko, Inc., NY).

Stroma cells that comprise fibroblasts, mesenchymal germ cells, reserve cells of the liver, neural germ cells, germ cells pancreatic and / or embryonic germ cells with or without other cells and elements described below are inoculated in the structure. Also, the cells found in connective tissue loose can be inoculated in the three-dimensional holder together with fibroblasts. Such cells include, but are not limited to, smooth muscle cells, endothelial cells, pericytes, macrophages, monoliths, plasma cells, mast cells, adiposites, etc. As previously explained, they can used fetal fibroblasts to form a matrix of "generic" three-dimensional stroma that will support the growth of a variety of different cells and / or tissues. Without However, a "specific" stroma tissue can be prepared by inoculation of the three-dimensional structure with fibroblasts derived from the same type of tissue to be cultured and / or to from a particular individual who will receive the cells and / or tissues grown in the crop according to the system of three dimensions of the invention.

Thus, in one embodiment of the invention, stromal cells that are specialized for the particular tissue can be cultured. For example, stromal cells of hematopoietic tissue, including, but not limited to, fibroblasts, endothelial cells, macrophages / monoliths, adiposites and reticular cells, can be used to form the sub-confluent stroma for long-term culture of the bone marrow in vitro . Hematopoietic stromal cells can be easily obtained from the "amateur coating" formed in bone marrow suspensions by centrifugation at low forces, eg, 3000 Xg. In the stroma layer that makes up the inner wall of the arteries, a high proportion of undifferentiated smooth muscle cells can be added to provide the elastic protein. Stromal cells of the liver may include fibroblasts, Kupffer cells, and vascular and endothelial cells of the bile duct. Similarly, glial cells can be used as the stroma to support the proliferation of neurological cells and tissues; glial cells for this purpose can be obtained by trypsinization or digestion of embryonic or adult brian collagenase (Ponten and Westermark, 1980, in S. Federof, L. Hertz, eds, "Advances in Cellular Neurobiology", Vol. 1, New Cork, Academia Press, pp. 209-227). The growth of cells in the culture of three-dimensional stromal cells can be further enhanced with the addition of the structure, or the coating of the support with proteins (e.g., collagens, elastic fibers, reticular fibers), glycoproteins, glycosaminoglycans (eg, heparan sulfate, 4-chondroitin sulfate, 6-chondroitin sulfate, dermatan sulfate, keratin sulfate, etc.), a cell matrix, and / or other materials.

In addition, germ cells of mesemchyme (assigned or unassigned lineage progenitor cells) are advantageous "stroma" cells for the inoculation of the structure. The cells can differentiate into osteocytes, fibroblasts of the tendons and ligaments, stromal cells of the marrow, adiposites and other connective tissue cells, chondriocytes, depending on the course, or endogenous growth or supplemented and regulatory factors and other factors including prostaglandins, interleukins and chalonas found from natural way that regulate proliferation and / or differentiation.

Fibroblasts can be easily isolated by disintegration of an appropriate organ or tissue that will serve as a source of fibroblasts. This can be easily achieved using techniques known to those skilled in the art. For example, the tissue or organ can be mechanically disintegrated and / or treated with digestive enzymes and / or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue in a suspension of individual cells without an appreciable breakage of cells. Enzymatic dissociation can be achieved by biting the tissue and treating the chopped tissue with any of the digestive enzymes either alone or in combination. This includes, but is not limited to, trypsin, chymotrypsin, collagenase, elastase, and / or hyaluronidase, DNase, pronase, dispase, etc. Mechanical breakage can also be achieved by a number of methods including, but not limited to, the use of crushers, mixers, sieves, homogenizers, pressure cells, or soundproofers, to name a few. For a review of tissue disintegration techniques, see Freshney, Culture of Animal Cells: A Manual of Basic Technique , 2d. Ed., AR Liss, Inc., New York, 1987, Ch. 9, pp. 107-126.

Once the tissue has been reduced to a suspension of individual cells, the suspension can be divided into sub-populations from which fibroblasts and / or other cells and / or stroma elements can be obtained. This can also be achieved using standard techniques for cell separation including, but not limited to, cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based on differential cell agglutinability in the mixed population. , conventional filtration and zone centrifugation, centrifugal decantation (counter-current centrifugation), gravity unit separation, counter current distribution, electrophoresis and fluorescence activated cell classification. For a review of clonal selection and cell separation techniques, see Freshney, Culture of Animal Cells: A Manual of Basic Techniques , 2nd Ed., AR Liss, Inc., New York, 1987, Ch. 11 and 12, p. . 137-168.

Fibroblast isolation can be carried out, for example, as follows: fresh tissue samples are vigorously washed and chopped in Hanks balanced salt solution (HBSS) in order to separate the serum. The chopped tissue is incubated for 1 to 12 hours in a freshly prepared solution of a dissociating enzyme such as trypsin. After said incubation, the dissociated cells are suspended, formed as small pellets or pellets by centrifugation and laminated in the culture plates. All fibroblasts will attack before other cells, so that the appropriate stromal cells can be isolated and grown selectively. The isolated fibroblasts can then be grown for confluence, stimulated from the confluent culture and inoculated into the three-dimensional matrix (see, Naughton et al., 1987, J. Med. 18 (3 and 4) 219-250). Inoculation of the three-dimensional structure with a high concentration of stroma cells, e.g., approximately 10 6 to 5 x 10 7 cells / ml, will result in the establishment of the three-dimensional stroma tissue in shorter periods of time.

After inoculation of the cells of stroma, the three-dimensional structure must be incubated in a appropriate nutrient medium. As previously mentioned, may be suitable for use, many means commercially affordable such as RPMI 1640, that of Fisher, of Iscove, of McCoy and the like. It is important that cell cultures of three-dimensional stroma be suspended or floated in the middle during the incubation period in order to maximize the proliferative activity The crop is "fed" periodically and the conditioned medium of the invention is recovered and processed as described below in the Sections 5.6 and 5.7. Thus, depending on the tissue to be cultivated. and of the desired types of collagen, the appropriate stromal cell (s) to inoculate the three dimensional matrix.

During the incubation of cell cultures of three-dimensional stroma, proliferating cells can be released from the womb These released cells can stick to the walls of the culture vessel where they can continue proliferating and forming a conclusive monolayer. This must be prevented or minimized, for example, by cell separation released during feeding, or by crop transfer from three-dimensional stroma to a new culture vessel. The presence of a conclusive monolayer in the vessel will "brake" the cell growth in the matrix and / or three-dimensional culture. The elimination of the conclusive monolayer or the transfer of culture to freshly prepared media in a new glass will restore  the proliferative activity of the culture system of three dimensions. It should be noted that the conditioned media of the invention are processed, if necessary, so that do not contain any complete cells (unless, naturally, whole cells are used for a specific application). Such deletion or transfer must be made in any culture vessel that has a monolayer of stroma that exceeds the confluence by 25%. Alternatively, the culture system must be agitated to prevent release of glued cells, or instead of the periodic feeding of the crops, the cultivation system can be established so that freshly prepared media flow continuously through the system. The flow rate can be adjusted so that maximize proliferation in three-dimensional cultivation, and and to wash the cells released from the culture by washing, so that Do not stick to the walls of the glass and grow to confluence.

Other cells, such as the cells of parenchyma, can be inoculated and grown in stroma tissue  I live in three dimensions.

5.4. Inoculation of Specific Tissue Cells in the Three Dimensional Stroma Matrix and Maintenance of Crops

Once the culture of stromal cells in three dimensions has reached the appropriate degree of growth, additional cells such as tissue-specific cells (parenchyma cells) or surface layer cells that are desirably cultivable can also be inoculated into live stroma tissue. The cells are grown in the stroma tissue alive in vitro to form a cultured counterpart of the native tissue and condition the media by making extracellular products in the media at similar ratios to physiological levels. A high concentration of cells in the inoculum will advantageously lead to increased proliferation in the culture much earlier than with low concentrations. The cells selected for inoculation will depend on the tissue to be cultured, which may include, but are not limited to, bone marrow, skin, liver, pancreas, kidney, neurological tissue, adrenal gland, mucosal epithelium, and smooth muscle, to name a few. Such cells with elaborate characteristic extracellular proteins such as certain growth factors in the media will result in optimized media for certain specific tissue applications.

For example, and not by way of limitation, you can cultivate a variety of epithelial cells in the tissue of live stroma of three dimensions. Examples of such cells epithelials include, but are not limited to, keratinocytes, Oral mucosa and gastrointestinal tract cells (G.I.). These epithelial cells can be isolated by enzymatic treatment of the tissue according to the methods known in the state of the technique, followed by expansion of these cells in the culture and application of epithelial cells to the matrix of cells three-dimensional stroma support. The presence of the support of stroma provides growth factors and other proteins that favor normal division and differentiation of cells epithelial

In general, the inoculum must include the cell "germinal" that has not been obtained from human embryo (also called the "reserve" cell) for said tissue; that is to say, those cells that generate new cells that mature in the specialized cells that form the different components of the tissue.

The parenchyma or other cells in the layer of surface used in inoculum can be obtained from of cell suspensions prepared by tissue disaggregation desired using standard techniques described to obtain stroma cells in the previous Section 5.3. The whole cell suspension itself to inoculate the tissue of live stroma of three dimensions. As a result, the cells regenerative contained in the homogenate will proliferate, mature, and they will be properly differentiated from the matrix, while the Non-regenerative cells will not. Alternatively, they can isolate particular cell types from fractions appropriate cell suspension using standard techniques described for fractionating stromal cells in the above Section 5.1 When cells can be easily isolated "germinals" or "reserve" cells, this can be used to preferentially inoculate the stroma support of three dimensions. For example, when bone marrow is cultured, the stromal of three dimensions can be inoculated with cells of bone marrow, both freshly prepared and derived from a sample cryo-preserved When the skin is grown, it can inoculate the three-dimensional stroma with melanocytes and keratinocytes When the liver is cultured, the stroma of three Dimensions can be inoculated with hepatocytes. When grown the pancreas, the three-dimensional stroma can be inoculated with endocrine pancreatic cells. For a review of the methods which can be used to obtain several parenchyma cells tissues, see, Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2nd Ed., A. R. Liss, Inc., New York, 1987, Ch. 20, p. 257-288.

In fact, different proportions of different types of collagen deposited in the stroma matrix before the inoculation can affect the growth of specific cells of tissues inoculated later. For example, for a growth optimal hematopoietic cells, the matrix must contain preferable form types III, IV and I of collagen in a ratio Approximately 6: 3.1 in the initial matrix. The proportions of types of collagen deposited can be manipulated or intensified to selection of fibroblasts that make the type of collagen appropriate. This can be achieved using antibodies. monoclonal of an appropriate subtype isotope that is capable of a activating complement, and defining the types of collagen private individuals These antibodies and supplements can be used to negatively select fibroblasts that They express the type of collagen desired. Alternatively, the stroma cells used to inoculate the matrix can be a mixture of cells that synthesize the appropriate type of collagen wanted. The distribution and origins of the various types of Collagen are shown in Table I.

TABLE I

Distributions and origins of various types of collagen Kind of collagen Main Distribution of Tissue Origin Cells I Ordinary connective tissue Fibroblasts and cells loosened and dense; fibers of collagen reticular; cells from muscle smooth Fibrocartilage Bone Osteoblast Dentine Odontoblasts II Hyaline and cartilage elastic Chondrocytes Vitreous body Of the eye Cells of the retina III Connective tissue loosened Fibroblasts and cells fibers reticular reticular Cap papillary of the dermis Glasses blood Cells muscle smooth; cells endothelial IV Membranes of base Epithelial cells Y endothelial Capsule of the lens of the Fibers of crystalline eye V Membranes fetal; placenta Fibroblasts Membranes of base Bones Muscle smooth Cells muscle smooth Fibroblasts SAW Tissue connective VII Membranes of base Fibroblasts, epithelial, fibrils clamped keratinocytes Cornea Fibroblasts from the cornea VIII Cartilage IX Cartilage hypertrophic X Cartilage Fibroblasts XI Dermis papillary Fibroblasts XII Dermis reticulate Fibroblasts XIV, Antigen pemfigoid buffo Keratinocytes undulina P170 XVII

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During incubation, the three-dimensional cell culture system must be suspended or floated in the nutrient medium. Crops should be fed with freshly prepared media, periodically. Again, care should be taken to prevent the release of cells from the culture of the glued on the vessel walls where they can proliferate and form a conclusive monolayer. The release of cells from the three-dimensional culture seems to occur more easily when diffuse tissues are cultured as opposed to structured tissues. For example, the three-dimensional skin culture of the invention is histologically and morphologically normal; The different layers of the dermis and epidermis do not release cells in the surrounding media. In contrast, the three-dimensional bone marrow cultures of the invention release mature non-adherent cells in the middle in the same manner in which said cells are released into the marrow in vivo . As previously explained, if the released cells stick to the culture vessel and form a conclusive monolayer, the three-dimensional culture proliferation will be "braked." This can be avoided by removing the cells released during feeding, transferring the three-dimensional culture to a new vessel, by agitating the culture to prevent the released cells from sticking to the walls of the vessel, or by the continuous flow of the freshly prepared media at a speed sufficient to replenish nutrients in the culture and separate the released cells. As previously mentioned, the conditioned media are processed, if necessary, so that they are free of whole cells and cell debris.

The growth and activity of cells in crop may be affected by a variety of factors of growth such as insulin, growth hormone, somatomedins, colony stimulating factors, erythropoietin, epidermal growth factor, factor hepatic erythropoietic (hepatopoietin), and growth factor of liver cells. Other factors that regulate proliferation and / or differentiation include prostaglandins, interleukins, and Chalonas of natural origin.

5.5. Genetic Engineering Constructions

In another embodiment, the constructions of three dimensions that condition the media can act as vehicles for the introduction of gene products in the media which, for example, favor the repair and / or regeneration of tissue defects The cells can be obtained by genetic engineering to express, for example, mediators inflammatory, such as IL-6, IL-8 and G-CSF. Alternatively, the cells too can be engineered to express anti-inflammatory factors, e.g. anti-GM-CSF, anti-TNF, anti-IL-1, anti-IL-2, etc.

In another embodiment, the cells can be obtained by genetic engineering to express a gene in the means that will exert a therapeutic effect, e.g. in the TGF-? production to stimulate cartilage production, or other factors such as BMP-13 to favor chondrogenesis or stimulate factors that favor stromal cell migration and (or the deposition of the matrix. Because the constructions comprise eukaryotic cells, the product of the gene It will be properly expressed and processed to form a product active. Preferably, the control elements of the Expression used should allow regulated gene expression so that the product can be over synthesized in the crop. The selected transcriptional promoter, in general, and the elements of the promoter specifically depend, in part, on the type of tissue and cultured cells. The cells and the tissues that are capable of secreting proteins are preferably (eg, those with abundant uneven endoplasmic reticulum and organelles of the Golgi complex). The product of the gene over-produced will then be segregated by Genetic engineering cells in conditioned media.

The cells used to condition the means can be engineered to regulate one or more genes; or the regulation of gene expression can be transient or long term; or gene activity can be inducible or non inducible.

The cells that condition the media can also be engineered to "suppress" the expression of factors that favor inflammation. Next, negative modulating techniques for reducing the expression levels of the target gene or the activity levels of the product of the target gene are discussed. "Negative modulation", as used in the present invention, refers to the reduction in the level and / or activity of the product of the target gene in relation to the level and / or activity of the product of the target gene in the absence of the modular treatment. The expression of a cell-native gene can be reduced or suppressed using a number of techniques, for example, expression can be inhibited by complete inactivation of the gene (commonly called "suppression") using standard homologous recombination techniques. Normally, an exon encoding an important region of the protein (or a 5 'exon for said region) is interrupted by a positive selectable marker (eg neo ), preventing the production of normal mRNA from the target gene and resulting in gene inactivation A gene can also be inactivated by creating a deletion or an inactivating insertion in part of a gene, or by deletion of the entire gene. By using a construct with two regions of homology to the target gene that far apart in the genome, the sequences involved in the two regions can be suppressed. Mombaerts et al. , 1991, Proc. Nat. Acad. Sci. USA 88 : 3084-3087. Alternatively, a gene can also be inactivated by suppression of countercurrent expression elements or in the same direction.

Ribozyme and antisense molecules that inhibit expression of the target gene can also be used in accordance with the invention to reduce the level of activity of the target gene. For example, antisense RNA molecules that inhibit the expression of the most histocompatible gene complexes (HLA) have been shown to be more versatile with respect to immune responses. In addition, appropriate ribozyme molecules can be designed as described, for example, by Haseloff et al. , 1988, Nature 334 : 585-591; Zaug et al., 1984, Science 224 : 574-578; and Zaug and Cech, 1986, Science 231 : 470-475. Still additionally, triple helix molecules can be used in reducing the level of activity of the target gene. These techniques are described in greater detail by LG Davis et al ., Editions, Basic Methods in Molecular Biology , 2nd ed., Appleton - Lange, Norwalk, Conn. 1994

The methods that may be useful for Genetic engineering cells of the invention are well known. in the state of the art and are described in greater detail in United States patents of co-holders, 4,963,489 and 5,785,964. For example, a DNA construct recombinant or a vector containing an exogenous nucleic acid, by example that encodes a product of the gene of interest, can be Built and used to transform or transfect cells of stroma of the invention. Said transformed cells or transfected that have exogenous nucleic acid, and that are capable of expressing said nucleic acid, are selected and expanded in cloned form in the constructions of three dimensions of this invention.

The methods for the preparation of DNA constructs containing the gene of interest, by transformation or transfection of cells, and by selection of cells that have and express the gene of interest are well known in the state of the art. See, for example, the techniques described in Maniatis et al. , 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY .; Ausubel et al., 1989, Current Protocols in Molecular Biology , Greene Publishing Associates - Wiley Interscience, NY; and Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring harbor, NY

The cells can be obtained by engineering genetics using any of a variety of vectors, including, but not limited to, integral viral vectors, by e.g., papillota virus vectors, SV40 vectors, vectors adenovirals; or viral vectors of defective replication. When a transient expression is desired, vectors may be preferred non-members and defective replication vectors, due to that both inducible and promoters can be used constitutive in these systems to control gene expression of interest. Alternatively, the integrating vectors can be used to obtain the transient expression, provided that The gene of interest is controlled by an inducible promoter. Others Methods of introducing DNA into cells include the use of liposomes, lipofection, electroporation, a trigger of particles, or by direct injection of DNA.

The cells are preferably transformed or transfected with a nucleic acid, e.g., DNA, controlled by, for example, in association with, one or more control elements of the appropriate expression such as promoter sequences or enhancers, transcription finalizers, sites of polyadenylation, among others, and a selectable marker. Following the introduction of foreign DNA, engineering cells  genetics can be grown in an enriched medium and to then changed to a selective medium. Marker selectable in foreign DNA confers resistance to selection and allows cells to stably integrate into DNA strange, such as, for example, in a plasmid, in its chromosomes and grow to form foci that, in turn, can be cloned and expanded in cell lines. This method can be advantageously used for engineering cell lines genetics that express the product of the gene in the media.

Any promoter can be used to direct the expression of the inserted gene. For example, viral promoters include but are not limited to the CMV promoter / enhancer, SV40, papillomavirus, Epstein-Barr virus, elastin gene promoter and β-globin. Preferably, the control elements used to control the expression of the gene of interest should allow regulated expression of the gene so that the product is synthesized only when required in vivo . If transient expression is desired, the constitutive promoters are preferably used in a non-integrating and / or defective replication vector. For example, inducible promoters include, but are not limited to, metallothioniene and heat shock protein.

According to one embodiment, the promoters inducible used for the expression of exogenous genes of interest are those who are the native promoters of those regulatory proteins as described herein document that are induced as a result of the cryopreservation and subsequent defrosting. For example, the TGF-? Promoter, VEGF, or Several known heat shock proteins can be used as the expression control element, that is, it can be operatively linked to an exogenous gene of interest in order to express a desired gene product in tissue constructs that condition cellular media.

A variety of methods can be used to obtain constitutive or transient expression of genetically engineered gene products in cells. For example, the transkaryotic implantation technique described by Seldon et al., 1987, Science 236 : 714-718 can be used. The term "transkaryotic," as used herein, suggests that the nuclei of the implanted cells have been altered by the addition of DNA sequences by stable and transient transfection. Preferably, the cells are genetically engineered to express said transient gene products and / or under inducible control during the post-operative recovery period, or as a chimeric fusion protein anchored to stromal cells, for example, as a chimeric molecule composed of an intracellular and / or transmembrane domain of a receptor or receptor type molecule, fused to the gene product as the extracellular domain.

In addition, it may be desirable to prepare a construction that has an extracellular matrix that contains a foreign gene product, a growth factor, a factor regulator, etc., which is found below in the media conditioned. This embodiment is based on the discovery of that, during the growth of human stroma cells in a three-dimensional support structure, the cells synthesize and deposit an extracellular matrix in the structure human as it occurs in normal human tissue. Matrix extracellular is secreted locally by cells and not only unites cells and tissues together but also influences the development and behavior of the cells in contact. Matrix Extracellular contains several connective tissue proteins, by eg, proteins that form inter-woven fibers in a hydrated gel composed of a structure of glycosaminoglycan chains. The glycosaminoglycans are a heterogeneous group of chains of long, negatively charged polysaccharides, which (except for the hyaluronic acid) are covalently bound to the protein to form proteoglycan molecules. According to this embodiment of the invention, stromal cells can be Genetically modified to express a product of desired gene, or altered forms of a gene product, which will be present in the extracellular matrix and finally the cellular medium.

5.6. Recovery of Conditioned Media

The cells can be cultured by anyone of the means known in the state of the art. Preferably the cells are grown in an environment that allows the Processing and aseptic manipulation. Conventional media of cell and tissue cultures have been limited by the need for human supervision and media control. This limits the amount of cells and tissues that can be cultured at the same time and consequently the volume of cellular media conditioners that can be obtained at the same time. For this reason, it prefers that the media be conditioned so as to allow a large-scale growth (yielding large-scale conditioned media scale) using, for example, an apparatus for aseptic culture  on a large scale as described in the patent of con co-ownership 5,763,267 (the '267 patent). Using the aseptic closed system described in the '267 patent, preconditioned culture media are transported from a fluid reserve to an inlet manifold and even distributed to crops in a continuous flow system and is useful in the culture of cells in three dimensions and in tissue cultures, such as Dermagraft® for example. In particular, the device described in the '267 patent includes a plurality of chambers of flexible or semi-flexible treatment that comprise one or more individual culture pockets, a plurality of rigid spacers, a fluid collector of inlet, an outlet fluid manifold, a fluid reservoir, and a means to transport the fluid within the system.

During the treatment, the liquid medium is transported from the fluid reservoir to the manifold of input, which in turn evenly distributes the media to each of the connected treatment chambers and to the pockets of internal cultivation. A fluid reserve of output to ensure that each treatment chamber is filled with uniformly and to ensure that any of the bubbles of air formed during treatment are removed from the chambers of treatment, The treatment chambers are flexible or semi-flexible so that they provide a ease of handling for the end user during rinsing and the application of cultivated transplants. Due to the flexibility of treatment chambers, are also provided rigid spacers that ensure the exact distribution of fluid inside the chambers during treatment. Where appropriate (that is, once the medium is conditioned so that the extracellular proteins such as growth factors have reached desirable levels in the middle) the middle "conditioning" is pumped out of the system and processed to its use. Preferably, the conditioned cell medium is collected of the apparatus in the last stages of tissue growth when the level of certain growth factors and the secretion of connective tissue protein is at its highest level (See Fig. one). In a preferred embodiment, the medium conditioned by the three-dimensional cell culture is collected after exposure of the medium to cells between days 10 and 14 of culture.

In another embodiment, the fabric of three dimensions is grown in an apparatus for aseptic growth of three-dimensional tissue cultures as described in U.S. Patent No. 5,843,766 (the '766 patent). The '766 patent describes a tissue culture chamber in which the chamber is a box that allows the tissue growth of three dimensions, preserved in a frozen form, and served at end user in the same aseptic container. The cultivation chamber of the fabric includes a box comprising a substrate within the box designed to facilitate tissue growth in three dimensions on the surface of the substrate. The box includes a port of entry and one of exit attending the entrance and the middle exit. The box also includes at least one distributor flow. In one embodiment, the flow distributor is a separator screen, which is used to distribute the flow of half inside the chamber to create a uniform, continuous piece of the three-dimensional fabric. In a second embodiment, the Flow distributor is a combination of baffle plates, distribution channels, and a flow channel. In each embodiment, the box also includes a closure so that an environment is ensured aseptic inside the chamber during the growth of the tissue and storage. Again the medium is preferably separated from the apparatus in the last stages of growth of the tissue when the level of secretion of growth factors and of Connective tissue protein is at its highest level (See Fig. 1). In a preferred embodiment, the medium conditioned by the culture Three-dimensional cell is collected after exposure from the medium to the cells between days 10 and 14 of the culture.

5.7. Concentration of the conditioned medium

After the cell-conditioned medium has been removed, it may be necessary to further elaborate the resulting supernatant. Although not limited thereto, such processing may include concentration by means of a filtration device with water fluidization or by filtration using the methods described in Cell & Tissue Culture; Laboratory Procedures, supra , pp. 29 D: 0.1-29D: 0.4.

Additionally, the medium can be concentrated increasing the concentration by 10 to 20 times using a positive pressure concentration device that has a 10,000 ml filter with cut-off point (Amicon, Beverly, MA).

Also, the conditioned medium can be further elaborated with a view to isolation and purification of the product, to remove unwanted proteases, for example. The methods that can be used for the isolation and purification of the product to maintain optimum biological activity will be completely obvious to one skilled in the art. For example, it may be desirable to purify a growth factor, a regulatory factor, a peptide hormone, an antibody, etc. Without being limited to these, such methods include ion exchange by gel chromatography (using matrices such as sephadex), affinity chromatography with metal chelates with an insoluble matrix such as cross-linked agarose, HPLC purification (HPLC = chromatography of high resolution liquids) and hydrophobic interaction chromatography of conditioned media. Such techniques are described in more detail in Cell & Tissue Culture; Laboratory Procedures, supra . Naturally, depending on the desired application of the conditioned medium and / or the products taken from it, appropriate measures must be taken to maintain sterility. As an alternative, sterilization may be necessary, and it may be carried out by methods known to a person skilled in the art, such as for example sterilization by heat and / or with filter, trying to preserve the desired biological activity.

5.7.1. Collagen Isolation

As mentioned above, the conditioned medium of the invention contains numerous products that can be isolated and purified therefrom. For example, human dermal fibroblasts synthesize and secrete collagen precursors, and a fraction of these precursors is incorporated into a three-dimensional extracellular matrix. This incorporation requires the removal of the terminal peptides (peptides N and C), which significantly reduces the solubility of the collagen molecules (the rest of the secreted collagen remains in solution due to lack of proteolysis). In general, soluble collagen can be obtained under neutral pH conditions at high salt concentrations. See Kielty, CM, I. Hopkinson et al. , (1993), Collagen: The Collagen Family: Structure, Assembly, and Organization in the Extracellular Matrix, Connective Tissue and Its Heritable Disorders: molecular, genetic and medical aspects . PM Royce and B. Steinmann. New York, Wiley-Liss, Inc .: 103-149. The Applicants provide data that demonstrate the effect of the conditioned medium (a medium that has previously supported the growth of cultured cells in three dimensions) in the preparation and composition of three-dimensional tissues by measuring the amount of collagen secreted into the extracellular matrix of cultured tissues in the presence of serum-free medium, medium or three-dimensional conditioned medium (see Part 6.3). The conditioned medium of the invention significantly increases collagen deposition of tissue in vitro as shown in Figure 4.

Applicants have also discovered that, surprisingly, the collagen is not deposited in a linear manner, but instead it is secreted at increasing levels during the cultivation process (see Figure 1). Consequently, the Applicants have applied this discovery by collecting the collagen

It should be understood that the following protocol is offered by way of example and can be modified using methods known to those skilled in the art. To purify the collagen, add 240 ml of fibroblast conditioned medium to 240 ml of 5 M NaCl (in a ratio of medium to salt of 1: 1), and precipitation for 16 hours at 4 ° Celsius. Centrifuge the suspension for approximately 20 minutes at 4000 g. Discard the supernatant. Wash the pellets with 10 ml of a solution of 50 mM Tris-HCl (pH 7.5) and 2.4 NaCl M. Centrifuge for 20 minutes at 4000 g and discard the supernatant Put the pellets back in suspension in 10 ml 0.5 M acetic acid To remove the propeptides, add 0.1 ml of pepsin (100 mg / ml) (Sigma Chemical, St. Louis, MO) and effect digestion for 16 hours at 4 ° Celsius (this removes the propeptides but leaves the triple helix intact). Centrifuge the suspension for 20 minutes at 4000 g. Retrieve the supernatant and discard pellets. Add 2.1 ml of 5M NaCl and 0.5 M acetic acid to a final volume of 15 ml (up to final NaCl concentration of 0.7 M). Precipitate by space of approximately 16 hours at 4º Celsius. Centrifuge the suspension for 20 minutes at 4000 g, and discard the supernatant Dissolve the pellets in 0.5 ml of acid solution 0.5 M acetic acid. Collagen purity should be at least 90% and can be analyzed by standard methods known in the technique such as that of the SDS-PAGE (SDS-PAGE = polyacrylamide gel electrophoresis with sodium dodecyl sulfate), for example.

5.8. Applications in which the Media are Used Conditioning 5.8.1. Wound Healing Applications

The conditioned media that are obtained by means of the invention can be developed to promote the wound and burn healing. When a tissue is injured, they are released into the wound to promote healing polypeptide growth factors that present a set of biological activities Wound healing is a process complex that involves several stages and is able to close the gaps of the integument in a controlled way to form tissue functionally competent The process begins with hemostasis, which it is followed by an inflammatory phase that involves neutrophils and macrophages The process continues with the development of tissue granulation and reepithelialization to close the wound. Then scar tissue is formed, and it is remodeled along the following months until an approach to the structure is achieved original anatomical Ideally, scar tissue is minimal, to that healthy tissue can be formed, that is functionally woven competent that histologically and physiologically resembles tissue normal original.

Each stage of the healing process is controlled by cellular interactions through regulatory proteins such such as cytokines, growth factors and inflammatory mediators, as well as intercellular contact mechanisms. For example, inflammatory mediators such as IL-6, the IL-8 (IL = interleukin) and the G-CSF (G-CSF = Stimulator Factor of Granulocyte colonies) induce lymphocyte differentiation and acute phase proteins, as well as infiltration, maturation and Neutrophil activation, which are processes that are important in the inflammatory stages of wound healing. Others examples of regulatory proteins involved in the process of wound healing are called VEGF (VEGF = Factor of Vascular Endothelial Growth), which induces angiogenesis during inflammation and the formation of granulation tissue, the BMP's (BMP's = bone morphogenic proteins), which induce bone formation, the so-called KGF (KGF = Growth Factor Keratinocyte), which activates keratinocytes, and the so-called TGF-? 1 (TGF = Growth Factor Transformant), which induces extracellular matrix deposition. In The (following) Table 2 gives a list of the concentrations of those of a series of growth factors that according to determination carried out by ELISA (ELISA = Enzyme-linked Immunoassay) are in the medium conditioned by the Applicants, who previously had sustained the growth of cultured cells in culture Dermagraft® tissue. It should be understood that the following list is not a list that completely includes all the factors and is given only in order to further characterize the medium conditioning indicating the concentration of some of the factors biologically active that are present in the middle of the invention.

TABLE 2

Concentrations of Growth Factors in the Conditioned Environment according to Measurement Made by ELISA VEGF 3.2 ng / ml G-CSF 2.3 ng / ml IL-8 0.9 ng / ml IL-8 3.2 ng / ml KGF 1.67 ng / ml TGF-? 0.8 ng / ml

In chronic wounds, the healing process is go interrupted at a point subsequent to hemostasis and before reepithelialization, and apparently it is seen in the inability to start over The inflammation seen in the wound is in mostly related to the infection, but the inflammation gives place to an environment rich in proteases that degrade proteins regulators and therefore interfere in the process of wound healing

Those of a variety of methods have been used to quantify and characterize the main molecular components that are secreted by the fibroblasts found in the three-dimensional tissue cultures TransCyte MF (MF = brand) and Dermagraft®. The matrix proteins and human glycosaminoglycans (GAGs) that are present in TransCyte MF and Dermagraft® cultures include, but are not limited to, collagen I and III, fibronectin, tenascin, decorin, veriscan, betaglycan and syndecane, as well. as other components (data not indicated). These proteins and these secreted GAGs are at the service of important structural functions and also stimulate the division, migration, adhesion and transduction of cellular signals. The deposition of glycosaminoglycans (the volume of deposition is dependent on the period of growth) and collagen (the volume of deposition is not dependent on the period of growth) in three-dimensional culture systems is illustrated in Figure 1. The components have been measured by ELISA, Western blot analysis, immunohistochemical analysis and PCR (PCR = Polymerase Chain Reaction test). For example, some of the components found in TransCyte MF include collagen I, III and VII (RNA) (RNA = ribonucleic acid), fibronectin, tenascin, thrombospondin 2, elastin, proteoglycans, decorin and versican, as well as other components (data not indicated). The activity of these components in the development, healing and normal functioning of tissues has been perfectly described. Additionally, Applicants describe certain effects of the human biomanipulated matrix on cell function in vitro . For example, Applicants have observed that cell proliferation is increased by the addition of biomanipulated matrix. To study its effects on cell proliferation, the matrix was physically removed from TransCyte MF and Dermagraft® cultures and was added in several dilutions to monolayer fibroblast cultures.
cough and human keratinocytes. The results of increased cell proliferation are illustrated in Figure 2.

In addition, as detailed in Part 6.3, the Applicants note that the effect of three-dimensional conditioned medium on the preparation and composition of three-dimensional tissues was examined by measuring the amount of collagen secreted into the extracellular matrix of tissues grown in the presence of free media. serum, medium or three-dimensional conditioned medium. The effect of the three-dimensional conditioned medium on the preparation and composition of three-dimensional tissues was examined by measuring the amount of collagen secreted into the extracellular matrix of cultured tissues in the presence of serum-free medium, medium or three-dimensional conditioned medium. The conditioned medium of the invention significantly increases the deposition of collagen from tissue in vitro as illustrated in Figure 4. The present invention contains many of the regulatory proteins that are thought to be important in wound healing and with respect to which It has been observed that they are depleted in in vivo wound healing models. In addition, in some medical states such as diabetes, the supply of some of the regulatory proteins that are necessary for wound healing is insufficient. For example, it has been discovered in a murine model of non-insulin-dependent diabetes (such as the mouse db / db) that VEGF and PDGF secretion (PDGF = Platelet-derived Growth Factor) and PDGF receptor expression they are all depressed in wounds compared to the levels that occur in the wounds of normal mice.

Also, the conditioned medium that is provided by the present invention is also useful in the treatment of other types of tissue damage, such as e.g. ex. those of a traumatic or congenital nature, in which repair and / or regeneration of tissue damage or defects is desired, since many of these growth factors are found in the conditioned cell media of the Applicants, including, for example, factors of fibroblast growth (FGFs), platelet-derived growth factors (PDGFs), epidermal growth factors (EGFs), bone morphogenetic proteins (BMPs) and transforming growth factors (TGFs), as well as those that modulate vascularization, such as vascular endothelial growth factor (VEGF), keratinocyte growth factor (KGF) and basic FGF, angiogenesis factors and antiangiogenesis factors. Stress proteins such as GR 78 and MSP90 induce growth factors such as TGF-?. TGF-?, Including TGF?-1, TGF?-2, TGF?-3, TGF?-4 and TGF?-5, regulate growth and differentiation and accelerate wound healing (Noda et al. 1989, Endocrin. 124: 2991-2995; Goey et al . 1989, J. Immunol. 143: 877-880, Mutoe et al . 1987, Science 237: 1333-1335). Mitogens such as PDGF increase the speed of cellularity and granulation in tissue formation (Kohler et al . 1974, Exp. Cell. Res. 87: 297-301). As mentioned above, cells are preferably human to minimize immunogenicity problems.

Due to the fact that the conditioned medium of the invention contains a set of such wound healing factors, the conditioned medium is advantageously used in the treatment for wound and burn healing, including skin wounds, fractures. of bones, gastric ulcers, lesions in the pancreas, in the liver, in the kidneys, in the spleen and in the blood vessels and other internal wounds. In addition, the conditioned medium can be combined with other medicinal ingredients such as antibiotics and analgesics. Embodiments include formulations of the conditioned medium with an ointment or ointment for topical applications. In fact, it has been shown that the conditioned medium of the invention induces the proliferation of human fibroblasts and keratinocytes. An increase in cellular response was observed by cells that were exposed to the conditioned medium for a period of time as short as that of 3 days in vitro (Figure 3).

Alternatively, the conditioned medium can be combined with a bandage (adhesive or non-adhesive) to promote and / or accelerate wound healing. The conditioned medium It can be used in any state, that is, it can be used in a liquid or solid state, having been lyophilized by freezing or drying in powder form or film form for topical wound treatments and applications non-stick, as well as injectable; see document PCT WO 96/39101.

As an alternative, the conditioned cellular medium of the present invention can be mixed with hydrogels polymerizable crosslinkers as described in U.S. Pat. No. 5,709,854, 5,516,532 and 5,654,381 and in WO 98/52543. Examples of materials that can be used for forming a hydrogel include modified alginates. Alginate it is a carbohydrate polymer that is isolated from seaweed and can be crosslinked to form a hydrogel by exposure to a divalent cation such as calcium, as described, for example, in WO 94/25080. Alginate is ionically crosslinked in the presence of divalent cations, in water, at temperature environment, to form a hydrogel matrix. In the sense in the which is used herein, the expression "alginates modified "refers to chemically modified alginates that They have modified hydrogel properties.

Additionally, polysaccharides that gel by exposure to monovalent cations, including bacterial polysaccharides such as gellan gum, and Vegetable polysaccharides such as carragaenins may be crosslinked to form a hydrogel using methods analogous to that are available for alginate crosslinking previously described.

Derivatives of modified hyaluronic acids. In the sense in which the used herein, the expression "hyaluronic acids" is refers to natural and chemically modified hyaluronic acids. Modified hyaluronic acids can be designed and synthesized with preselected chemical modifications to adjust the speed and degree of crosslinking and biodegradation

Hydrogel precursors are also useful covalently crosslinkable. For example, a polyamine soluble in water, such as chitosan, can be crosslinked with a water soluble diisothiocyanate such as diisothiocyanate polyethylene glycol

Alternatively, polymers that include substituents that are crosslinked by means of a radical reaction can be used when contacting a radical initiator. For example, polymers can be used that include ethylenically unsaturated groups that can be photochemically crosslinked, as described in WO 93/17669. In this embodiment, water-soluble macromers are provided that include at least one water-soluble zone, a biodegradable zone and at least two free radical polymerizable zones. Examples of these macromers are polyethylene glycol oligolactylacrylates in which acrylate groups are polymerized using radical initiator systems such as an eosin dye, or by brief exposure to ultraviolet light or visible light. Additionally, water-soluble polymers that include cinnamoyl groups that can be photochemically crosslinked can be used, as described in Matsuda et al ., ASAID Trans ., 38 : 154-157 (1992).

Preferred polymerizable groups are acrylates, diacrylates, oligoacrylates, dimethacrylates, oligomethacrylates and other biologically photopolymerizable groups acceptable. Acrylates are the active group species more preferred polymerizable.

The naturally occurring polymers and the synthetic polymers can be modified using reactions Chemicals available in the art and described, for example, in March, "Advanced Organic Chemistry", 4th Edition, 1992, Wiley-Interscience Publication, New York

The polymerization is preferably initiated using photoinitiators Useful photoinitiators are those that can be used to initiate polymerization of macromers no cytotoxicity and within a short space of minutes at most and with the maximum preference of seconds.

They can be used for light curing Numerous dyes Suitable dyes are perfectly known to those skilled in the art. Preferred dyes include erythrosine, floxin, rose bengal, tonina, camforquinone, ethyl eosin, eosin, methylene blue, riboflavin, the 2,2-dimethyl-2-phenylacetophenone, the 2-methoxy-2-phenylacetophenone, the 2,2-dimethoxy-2-phenylacetophenone, other derivatives of acetophenone and camforquinone. The Suitable cocatalysts include amines such as N-Methyldiethanolamine, N, N-dimethylbenzylamine, triethanolamine, triethylamine, dibenzylamine, N-benzylethanolamine e isopropylbenzylamine. Triethanolamine is a cocatalyst favorite.

In another embodiment, the conditioned medium obtained by the invention, or alternatively certain extracellular matrix proteins made in the medium, are used to have an excellent substance to cover sutures. The naturally secreted extracellular matrix provides the type I and type III collagen conditioning medium, fibronectin, terascin, glycosaminoglycans, acidic and basic FGF, TGF-? And TGF-?, KGF, versican, decorin and several other dermal matrix proteins Human secreted. Similarly, cellular media conditioning of the invention or matrix proteins extracellular removed from conditioned media can be used to coat conventional implantation devices, including vascular prostheses, in surgical accesses for correct defects in the body, resulting in obtaining superior implantation devices. The implants must be made of biocompatible inert materials that replace or replace defective function, and should be made of non-biodegradable materials or biodegradable materials. By covering the implantation devices with the medium that contains these extracellular proteins, the implant propitiates the correct cell fixings, resulting in the appearance of superior quality tissue at the implantation site. Therefore, sutures, bandages and implants coated with conditioned cellular medium or with proteins removed of the environment increase the recruitment of cells such as leukocytes and fibroblasts inside the injured area and induce cell proliferation and differentiation, resulting in a Enhanced wound healing.

In another embodiment, the conditioned medium can be mixed with a pharmaceutically acceptable carrier as vehicle for internal administration. Also, the medium can be additionally developed to concentrate or reduce one or more factors or components that are contained within the environment, for example, an enrichment can be made in a factor of growth using immunoaffinity chromatography, or being able to conversely, removal of a less desirable component for any given application of those here describe.

Naturally, wounds in tissues specialized may require a conditioned medium for that fabric  specialized. For example, neuronal tissue lesions may require proteins contained in a conditioned medium by neuronal cell cultures. Products can be obtained specific, or alternatively the conditioned medium can be enriched by immunoaffinity chromatography or by an increased expression of a desired protein from the medium specific, such as the case of NGF (NGF = Factor of Neuronal Growth). Although not limited to these, the features that are controlled by the NGF include the function of cholinergic neurotransmitters (acetylcholinesterase (AChE) and acetylcholine synthesis enzyme (ChAT)), size of neuronal cells and the expression of NGF receptors Type II; the NGF being secreted into the middle conditioning by glial cells and other neuronal cells cultured in a three-dimensional stromal tissue, being able to be then used in a composition for the healing of nerves.

Endogenous NGF deficits aggravate certain human neurodegenerative disorders, and there is an apparent incapacity of neurons of the adult central nervous system Injured to regenerate. Specifically, a nerve injury is followed by a degeneration of nerve fibers in a distal location with respect to the lesion, the result being of axon isolation with respect to the cell body. At central nervous system, there is no significant growth in the site of injury, which typically leads to death of the damaged neuron The NGF plays a crucial role in capabilities Regenerative cholinergic neurons of the nervous system adult central at the level of the cell body (eg of the septum), in intermediate tissue spaces (such as the nervous bridge) and in the reinnervation zone (eg the formation hippocampus). Additionally, the NGF can be beneficial for Improve cognitive defects. The medium conditioned with cells glials, for example, can supply exogenous NGF and other factors of nerve growth so that from the cut ends of the Injured nerve can grow new axons (developing e.g. a growth cone) that extend to the original site of the connection.

In addition, brain and spinal cord injury is often accompanied by a glial response to concomitant axonal degeneration, resulting in scar tissue. Initially it was thought that this scar tissue constituted a physical barrier to nerve growth, although the presence or absence of neuronotropic factors in the extraneuronal environment is of greater importance. Astrocytes appear to be able to synthesize laminin in response to a lesion (laminin can also be found in the conditioned medium as discussed in more detail in Part 5.8.2 regarding extracellular matrix proteins). It has been found that collagen and fibronectin, and especially laminin, promote the growth of neurites from neurons or neuronal explants grown in vitro . These extracellular matrix proteins appear to provide an adhesive substrate that facilitates the advance movement of the growth cone and axon elongation. Thus, for a successful nerve regeneration the presence of neuronotropic factors and a sustaining substrate is necessary, since regeneration seems to require that the neuronal cell body be able to mount the appropriate biosynthetic response and that the environment surrounding the site of the lesion be able to support the lengthening and finally the functional reconnection of the axon. The medium conditioned by nerve cells such as astrocytes and glial cells contains the neuronotropic growth factors and extracellular matrix proteins that are necessary for nerve regeneration in brain and spinal cord lesions.
Thus, in one embodiment the conditioned medium is incorporated into formulations for the treatment of such lesions.

In other embodiments, the treatment of skin, bones, liver, pancreas, cartilage and of other specialized fabrics can be carried out with means conditioned by their respective types of specialized cells, preferably cultivated in three dimensions, resulting in a conditioned medium containing extracellular proteins characteristics and other metabolites of that type of tissue that are useful for treating wounds inflicted on that respective type of tissue.

The conditioned cellular medium can also be added to devices used in periodontal surgery in order to promote uniform tissue repair, to make grafts bone, corneal shields or biodegradable contact lenses, for make fillings of surgical spaces, to promote the increase of soft tissue, particularly in the skin in order to reduce the skin wrinkles, and for the increase of the urinary sphincter, in order to control incontinence

In another embodiment, the compositions may be freeze-dried / freeze dried and added as filler wounds (eg to fill the holes left by the plugs capillaries for implantation), or they can be added to existing wound filling compositions to accelerate the wound healing In another embodiment, the medium is conditioning with genetically engineered cells to increase The concentration of wound healing proteins in the environment. For example, cells can be manipulated to express gene products such as any of the factors of growth that have been listed above.

5.8.2. Repair and Correction of Abnormalities Congenital, Acquired Defects and Cosmetic Defects

Media compositions can also be used to repair and correct a variety of anomalies both congenital as acquired, as well as cosmetic defects both superficial as invasive. For example, the compositions may be added in any form and can be used in a hydrogel, an injectable, a cream or an ointment, and can even be added to an eye shadow, a compact makeup, to compact powders or other cosmetics to strengthen the skin topically

In another embodiment topical application is performed or application by any known method such as that of injection, oral administration, etc. of the conditioned medium to reverse and / or prevent wrinkles and a series of effects harmful induced by ultraviolet light, exposure to a variety of pollutants and normal aging, by example.

Additionally, in another embodiment the means of the invention is used to reduce cell aging and inhibit the activity of the factors that cause skin cancer. It is shown in Part 7.1 that the conditioned medium has antioxidant activity Again, application to a mammal can be topical or an application made by any method known as injection, oral administration, etc. The Applicants have discovered that in human keratinocytes exposed to the conditioned medium of the Applicants was observed a statistically significant reduction (p <0.003) of the intracellular oxidation of approximately 50 percent.

Thus, in addition to inducing epidermal and dermal cell proliferation and collagen secretion in vitro , the conditioned medium of the invention has a strong antioxidant activity (Fig. 5). Likewise, the factors are relatively stable, and the contents of TGF? 1, VEGF and collagen were stable after 21 days of storage at 37 ° C at a pH of 7.4 and 5.5. The solutions stored 2 + years at -20 ° C maintained stable levels of TGFβ1 and VEGF.

This sterile enriched nutrient solution represents a biomanipulated cosmeceutical that is easily available in large volumes and can be useful as an additive for a variety of dermatological, cosmetic and skin products to supplement the levels of growth factors and molecules of matrix in human nails, hair and skin. Is contemplated the use of products with Alfa Hydroxy Acid scrubs for potentially optimize the penetration of the factors of growth and other biomolecules inside the skin and with chemical scrubs to potentially accelerate healing and reduce inflammation

The conditioned medium can be incorporated into a formulation to eliminate wrinkles, frown marks, scar tissue formation and other skin conditions instead of using silicone or other products to do so. The conditioned medium contains growth factors and inflammatory mediators such as, for example, VEGF, HGF (HGF = Hepatocyte Growth Factor), IL-6 (IL = interleukin), IL-8, G-CSF ( G-CSF = Granulocyte Colony Stimulating Factor) and TGF? 1 (see Table 3 in Part 5.8.1), as well as extracellular matrix proteins such as type I and type III collagen, fibronectin, tenascin, glycosaminoglycans, acidic and basic FGF, TGF-? and TGF-?, KGF, versican, decorin, betaglycans, syndecane and various other secreted human dermal matrix proteins They are useful for repairing physical anomalies and cosmetic defects. As detailed in Part 6.3, the Applicants note that the effect of three-dimensional conditioned medium on the preparation and composition of three-dimensional tissues was examined by measuring the amount of collagen secreted into the extracellular matrix of tissues grown in the presence of serum-free media. , half or half three-dimensional conditioning. The conditioned medium of the invention significantly increased the deposition of collagen from tissue in vitro as shown in Figure 4. The effect of the three-dimensional conditioned medium on the preparation and composition of three-dimensional tissues was examined by measuring the amount of collagen secreted into the interior of the extracellular matrix of tissues grown in the presence of serum-free medium, medium or three-dimensional conditioned medium. Naturally, the cells used to condition the medium can be genetically manipulated to express improved concentrations of such proteins in the medium.

The conditioned medium that is obtained by the invention can be used in formulations to make injectable preparations. As an alternative, products taken from the conditioned medium can be incorporated into the formulations. For example, biologically active substances such as proteins and drugs can be incorporated into the compositions of the present invention for the release or controlled release of these active substances upon injection of the composition. Examples of biologically active substances may include tissue growth factors such as TGF-? And similar factors that promote tissue healing and repair at the injection site. Although not limited to these, product purification methods include gel chromatography using matrices such as SEPHADEX®, ion exchange, affinity chromatography for metal chelates, with an insoluble matrix such as cross-linked agarose, HPLC purification and hydrophobic interaction chromatography of the conditioned medium. Such techniques are described in more detail in Cell & Tissue Culture; Laboratory Procedures, supra ; Sanbrook et al ., 1989, Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Lab Press, Cold Spring Harbor, NY.

In the injectable embodiment a aqueous suspension and the aqueous suspension formulation will have typically a physiological pH (ie, a pH of approximately 6.8 to 7.5). Additionally, an anesthetic is usually added local such as lidocaine (usually at a concentration of approximately 0.3% by weight) to reduce local pain after the injection has been made. The final formulation too It will typically contain a fluid lubricant such as maltose, which It must be tolerated by the body. The component examples Lubricants include glycerol, glycogen, maltose and similar components. They can also act as lubricants base materials that are organic polymers such as polyethylene glycol and hyaluronic acid, as well as non-collagen fibrillar, and preferably succinylated collagen. Such lubricants are generally used to improve injectability, the penetrability and dispersion of the biomaterial injected into the injection site and to reduce the amount of obstruction modifying the viscosity of the compositions. Formulation final is by definition the conditioned cellular medium developed in a pharmaceutically acceptable carrier.

The prepared air conditioning is subsequently placed in a syringe or other injection device to accurately locate the air conditioner at the site of the tissue defect In the case of formulations for increase dermal, the word "injectable" means that the formulation it can be administered from syringes that have such a caliber Small as 25 under normal conditions and under pressure normal without considerable obstruction. Obstruction can do that the composition oozes from the syringe instead of being injected inside the fabric. For this precise location they are desirable needles as thin as those of 27 gauge (ID 200 µ) or even those of caliber 30 (ID of 150). The maximum size of particles that can be ejected by such needles will be a complex function of at least the following factors: the maximum particle size, particle shape ratio (length: width), particle stiffness, roughness surface of particles and related factors that affect the adhesion of particle with particle, the properties viscoelastic suspension fluid and flow rate of needle circulation. Rigid spherical pearls in suspension in a Newtonian fluid represent the simplest case,  while it is more likely that the case of fibrous or branched particles in a fluid viscoelastic

The steps described above which are carried out in the process of preparing an injectable secreted human conditioned medium are preferably carried out under sterile conditions and using sterile materials. The conditioned medium made in a pharmaceutically acceptable carrier can be injected intradermally or subcutaneously to increase soft tissue or to repair or correct congenital abnormalities, acquired defects or cosmetic defects. Examples of such conditions are congenital anomalies such as hemifacial microsomy, malar and zygomatic hypoplasia, unilateral breast hypoplasia, pectus excavatum , pectoral agenesis (Poland anomaly) and velopharyngeal incompetence secondary to cracked palate or palate repair submucosal fissure (as a retropharyngeal implant); acquired defects (post-traumatic, post-surgical, post-infectious) such as depressed scars, subcutaneous atrophy (eg secondary to discoid lupus erythematosus), keratotic lesions, enophthalmia in the sunken eye (also upper sulcus syndrome), acne rash on the face , linear scleroderma with subcutaneous atrophy, saddle nose deformity, Romberg disease and unilateral vocal cord paralysis; and cosmetic defects such as glabellar frown lines, deep nasolabial wrinkles, circumoral geographic wrinkles, sunken cheeks and breast hypoplasia. The compositions of the present invention can also be injected into internal tissues such as the tissues that define the body sphincters, to augment such tissues.

Other types of tissues that are used to conditioning the medium include, but not limited to, the bone marrow, skin, epithelial cells and cartilage; yes it is well understood that the culture system three-dimensional can be used with other types of cells and tissues.

As an alternative, the conditioned cellular medium which is obtained by the present invention can be mixed with polymerizable or crosslinking hydrogels as described in the previous part about wound treatment.

5.8.3. Food Additives and Supplements Dietetics

The conditioned media can be used in quality of food additives and can be incorporated into Dietary supplement formulations. The medium that is obtained by the invention contains many useful nutrients which include essential amino acids, minerals and vitamins in an abundance and variety that are not given in individual groceries or in groups of groceries. The Applicants are not knowledgeable about a food item anymore balanced (apart from breast milk) containing a tan extensive set of nutrients, although they are made such attempts at expensive liquid formulas of special formulation that They are available for both adults and babies. The media conditioned cell phones and / or products taken from them can be used as an economical source of nutritional supplement balanced for weight loss or as an alternative to increase the nutritional content of edibles, particularly for third world countries. The medium is sterile and It is free from contamination by human pathogens (ie it is aseptic). The conditioned medium can be concentrated and / or lyophilized, and can be preferably administered in capsules or tablets for ingestion. Alternatively, the compositions can be directly added to groceries for adults or babies to increase the nutritional content. This rich source of nutrients can be made relatively inexpensively and It can be invaluable for undernourished people of age advanced and particularly for children in countries underdeveloped, where it has been associated with the malnutrition increased mortality due to poor Infection responses.

Additionally, many of the trace elements found in conditioned media, such as iron and magnesium, are decisive for the survival and reproduction of mammals, and the issue of marginal trace element deficiency may be a problem. of public health. It has been suggested that ingestion of several essential micronutrients reduces the risk of infection as well as the risk of cancer by modifying specific phases of carcinogenesis. Micronutrients also increase the functional activities of the immune system and its mechanism of interaction of T cells and B cells, M diameters and NK cells specifically based on increasing the production of several cytokines to facilitate their phagocytic and cytotoxic action against invading pathogens and / or to destroy emerging premalignant cells in several vital organs. See Chandra, RK ed. (1988), Nutrition and Immunology: Contemporary Issues in Clinical Nutrition , Alan R. Liss, New York. Therefore, there is a need for a relatively inexpensive source of balanced nutrients. The food products that are ideal to be enriched with conditioned media are breads, breakfast cereals and other cereal products such as pastries, cookies, etc. Likewise, the medium can be further elaborated to concentrate or reduce one or more of the factors or components that are contained in the medium, for example, enrichment can be made in a growth factor using immunoaffinity chromatography, or the reverse can be done a removal of a less desirable component, for any given application than those described in this Part.

5.8.4. Feed Supplement

The compositions can be used as feed supplements. In one embodiment, the conditioned medium  it contains bovine serum, which constitutes a source of protein and other factors that are beneficial to mammals such as the cattle and other ruminant animals such as cows, deer and similar animals. The medium is selected for ensure the absence of pathogens, and is free of mycoplasma and bovine pathogens. The conditioned medium that is obtained by the invention is preferably obtained from cows raised in the United States, which significantly decreases the probability that they are present in the same pathogens.

5.8.5. The Cell Culture Medium

The compositions constituting the means can be "reused" to grow cells, and in particular cells that are difficult to grow in vitro . The conventional culture medium is typically supplemented with many of the factors that are already present in the Applicants' conditioned medium. In addition, the conditioned medium also contains factors that promote cell fixation and growth, such as extracellular matrix proteins that have been described above. Increasing fibronectin or collagen concentrations may be beneficial to promote the fixation of cells to a scaffold or culture surface. Instead of adding these factors to the medium, the conditioned medium can be used to grow cells and prepare three-dimensional tissue constructs such as Dermagraft®. Applicants have shown that the conditioned medium increases cell proliferation of fibroblasts and keratinocytes; see Figure 3. Cellular debris or other particulate matter, as well as proteases, lactic acid and other components that may be detrimental to cell growth can be removed from the medium before reuse as a cell culture medium. It may also be desirable to use serum in the conditioned cell medium for this application. The serum also contains binding factors such as fibronectin and serum diffusion factor, which promote the fixation of the cells to the substrate. Such fixation is necessary for the growth of some cells, but not all, in vitro . In addition to providing substances that are necessary for cell growth, serum can also play a role in stabilizing and detoxifying the culture environment. For example, the serum has an important buffering capacity and contains specific protease inhibitors such as α 1 -antitrypsin and α 2 -macroglubulin. Whey albumin, which is present at high levels in a medium that contains for example 10% serum, can act as a nonspecific inhibitor of proteolysis, as well as fix fat-soluble vitamins and steroid hormones that can be toxic in their free forms. The serum components can also fix and detoxify heavy metals and reactive organic compounds that may be present in the components of the medium.

5.8.6. Pharmaceutical Applications

The conditioned medium that is obtained by the invention contains a variety of useful pharmaceutical components and factors such as growth factors, regulatory factors, peptide hormones, antibodies, etc., as described throughout the specification, and is therefore Useful for a variety of pharmaceutical applications. Also, although not limited to these, the products that can be added include antibiotics, antivirals, antifungals, steroids, analgesics, antitumor drugs, research drugs or any compounds that result in a complementary or synergistic combination with the factors of the conditioned medium. As stated above, the cells are cultured, and the media are recovered under aseptic conditions. Additionally, the media can be analyzed to detect the presence of pathogens. If sterilization is done, it should be performed in a way that minimally affects the desired biological activity, as described above. The medium can be further elaborated to concentrate or reduce one or more of the factors or components that are contained in the medium, for example enrichment can be made in a growth factor using immunoaffinity chromatography, or the reverse being able to remove one less component desired for any given application described here. In a preferred embodiment, the formulations are made based on conditioned medium by a three-dimensional cellular construct. Three-dimensional cultures produce a multitude of growth factors and proteins that are secreted to the environment in optimal proportions and physiological concentrations. See, for example, Table 2 in Part 5.8.1. The medium therefore provides a unique combination of specified factors and proportions that closely represent those that occur in vivo . Bovine serum is not generally preferred in this application. It may be preferable to remove cellular debris or certain other materials, as well as proteases, lactic acid and other components that may be detrimental to cell growth.

They can be done with the conditioned medium Pharmaceutical products made in the form of tablets, capsules, skin patches, inhalers, eye drops, eye drops the nose, ear drops, suppositories, creams, ointments, injectables, hydrogels and any other appropriate formulation of those that are known to a person skilled in the art. For oral administration pharmaceutical compositions may adopt the form of for example tablets or capsules prepared by conventional media with pharmaceutically acceptable excipients such as binding agents (eg corn starch pregelatinized, polyvinylpyrrolidone or hydroxypropyl methylcellulose), fillers (eg lactose, cellulose microcrystalline or calcium and hydrogen phosphate), lubricants (such as p. ex. magnesium stearate, talc or silica), disintegrants (such as p. ex. potato starch or sodium starch glycolate), or agents wetting agents (such as sodium lauryl sulfate). Tablets can be coated using methods perfectly known in the technique. Liquid preparations for oral administration may take the form of for example solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or another suitable vehicle before use. Such liquid preparations can be prepared by means. conventional with pharmaceutically acceptable additives such as suspending agents (eg cellulosic syrup derivatives of sorbitol or hydrogenated edible fats), agents emulsifiers (eg lecithin or acacia), non-aqueous vehicles (such as almond oil, oily esters, ethyl alcohol or fractionated vegetable oils) and preservatives (such as methyl or propyl-p-hydroxybenzoates or acid sorbic). The preparations may also contain buffer salts and flavoring, coloring and sweetening agents as appropriate.

The pharmaceutical formulations of the invention can be administered to a patient by a variety of routes using standard procedures of those that are perfectly known to those skilled in the art. For example, such administration can be an oral, nasal administration, intravenous, subcutaneous, intradermal, transdermal, intramuscular or site specific intraperitoneal. Also said formulations can be formulated to work as slow-release controlled vehicles.

Although not limited to these, the therapeutic products contained in conditioned media include enzymes, hormones, cytokines, antigens, antibodies, coagulant factors and regulatory proteins. Although not left limited to these, therapeutic proteins include mediators inflammatory factors, angiogenic factors, Factor VIII, Factor IX, erythropoietin (EPO), alpha-1 antitrypsin, calcitonin, glucocerebrosidase, human growth hormone and derivatives, low density lipoprotein (LDL) and apolipoprotein E, IL-2 receptor and its antagonists, insulin, globin, immunoglobulins, catalytic antibodies, interleukins (ILs), insulin-like growth factors, superoxide dismutase, immune response modifiers, BMPs (bone morphogenic proteins), parathyroid hormone and interferon, nerve growth factors, plasminogen activators tissue and colony stimulating factors (CSFs). Naturally, the medium can be further elaborated to concentrate or reduce one or more of the factors or components that are contained in the medium, for example, a enrichment in a growth factor using chromatography  of immunoaffinity, or being able to carry out a removal of a less desirable component, for any application determined from those described here.

Analysis of what they are can be used commonly used by experts in the field to verify the activity of the specific factor or of the specific factors, thereby ensuring that the fixed molecule or the molecule encapsulated retain an acceptable level of biological activity (such as p. ex. a therapeutically effective activity).

Thus, conditioned cellular media and products taken from the means obtained by the invention they can be used, for example, to provide insulin in the Diabetes treatment, nerve growth factor for treatment of Alzheimer's disease, factor VIII and others coagulant factors for the treatment of hemophilia, dopamine for the treatment of Parkinson's disease, encephalin to through adrenal chromaffin cells for the treatment of chronic pain, dystrophin for the treatment of dystrophy muscle and human growth hormone for the treatment of abnormal growth

The doses of such protein agents Therapeutics are perfectly known to experts in the matter and can be found in pharmaceutical summaries such as PHYSICIANS DESK REFERENCE, Medical Economics Data Publishers; REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co .; GOODMAN & GILMAN, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, McGraw Hill Publ., THE CHEMOTHERAPY SOURCE BOOK, Williams and Wilkens Publishers

The therapeutically effective doses of any of the drugs or agents that have been described above can be routinely determined using techniques that are perfectly known to those skilled in the art. The term "therapeutically effective dose" refers to that amount of the compound that is sufficient to result in an improvement of at least one symptom of the processes and / or diseases that are treated.
so.

The toxicity and therapeutic efficacy of drugs can be determined by pharmaceutical procedures standard in cell cultures or experimental animals, e.g. ex. to determine the LD50 (the lethal dose for 50% of the population) and ED 50 (the therapeutically effective dose in the 50% of the population). The relationship between the corresponding doses To the toxic and therapeutic effects is the therapeutic index, and the It can be expressed as the ratio LD 50 / ED 50. Be prefer compounds that have large rates therapeutic While compounds with effects can be used toxic side effects, we must try to design a system of supply that directs such compounds to the tissue site affected in order to minimize potential damage to non-cells infected and therefore reduce the effects secondary.

The data obtained from the analyzes performed by cell culture and animal studies can be used to formulate a dosage range for use in humans The dosage of such compounds will be preferably located within a range of concentrations circulars that include the ED_ {50} with little or no toxicity. The dosage may vary within this range of values depending on the dosage form used and of the administration path used. For all compound used in the method of the invention, the therapeutically effective dose It can be estimated initially from tests performed by cell culture. A range is determined in cell culture of circulating plasma concentrations that include the IC50 (i.e. the concentration of the test compound that achieves a semi-maximal inhibition of symptoms). Such information can be used to more accurately determine the useful doses in the humans. Plasma levels can be measured, for example, by High performance liquid chromatography.

Additionally, cells and tissues can genetically manipulated to increase the expression of a desired product such as insulin, for example, and / or to express nucleotide sequences and / or halves that aim at gene products listed above, such as p. ex. the ribocime, antisense molecules and triple helices, which may have an inhibitory effect on gene expression and / or activity objective. This could be advantageous when growing tissues in which specialized stromal cells in the middle can play specific structural / functional roles, such as p. ex. glial cells of neurological tissue, the cells of Kupffer of the liver, etc.

5.8.7. Stimulation of Hair Growth

The medium can be conditioned using human capillary papilla cells, for example. Preferably, the medium conditioned by such cells is cultured in three dimensions. Capillary hair cells are a type of mesenchymal stem cell that plays a central role in the formation, growth and restoration of hair (Matsuzaki et al., Wound Repair Regen , 6: 524-530 (1998)). The conditioned medium is preferably concentrated and applied as a topical formulation. The compositions of conditioned media can be formulated for topical applications using an agent that facilitates the penetration of the compound into the skin, such as DMSO (DMSO = dimethyl sulfoxide), and can be applied in a topical application regime to stimulate growth hair related.

The compositions that are obtained by the invention promote or restore hair growth when applied topically, providing growth factors and other factors that increase the migration of epithelial cells to the hair follicles. In addition to the growth factors found in conditioned media, other compounds such as minoxidil and antibiotics can be used. During hair growth there is a reduction in blood supply during the catagen (the transition phase of the hair follicle between the growth and resting phases) and the telogen (the resting phase). Biologically active molecules taken out of the conditioned cellular medium can be determined and optimized for use during these hair growth phases using tests known in the art, including that performed with the short-tailed macaque model for baldness. male distribution; see, for example, Brigham, Pa, A. Cappas, and H. Uno, The Stumptailed Macaque as a Model for Androgenetic Alopecia: Effects of Topical Minoxidil Analyzed by Use of the Folliculogram, Clin Dermatol , 1988, 6 (4): p . 177-87; Diani, AR and CJ Mills, Immunocytochemical Localization of Androgen Receptors in the Scalp of the Stumptail Macaque Monkey, a Model of Androgenetic Alopecia, J Invest Dermatol , 1994, 102 (4): p. 511-4; Holland, JM, Animal Models of Alopecia, Clin Dermatol , 1988, 6 (4): p. 159-162; Pan, HJ, et al ., Evaluation of RU58841 as an Anti-Androgen in Prostate PC3 Cells and a Topical Anti-Alopecia Agent in the Bald Scalp of Stumptailed Macaques, Endrocrine , 1998, 9 (1): p. 39-43; Rittmaster, RS, et al. , The Effects of N, N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide, a 5 alpha-reductase Inhibitor and Antiandrogen, on the Development of Baldness in the Stumpatail Macaque , J. Clin Endrocrinol Metab , 1987, 65 (1): p. 188-93. Additional models include the measurement of differences in the proliferation of hair follicles from cultured follicles of bald areas and areas with hair, a neonata rat model, as well as a alopecia areata rat model; see Neste, DV, The Growth of Human hair in Nude Mice, Dermatol Clin ., 1996, 14 (4): p. 609-17; McElwee, KJ, EM Spiers, and RF Oliver, In Vivo Depletion of CD8 + T Cells Restores Hair Growth in the DEBR Model for Alopecia Areata, Br J Dermatol , 1996, 135 (2): p. 211-7; Hussein, AM, Protection Against Cytosine Arabinowide-Induced Alopecia by Minoxidil in a Rat Animal Model, Int J Dermatol, 1995, 34 (7): p. 470-3; Oliver, RF, et al. , The DEBR Rat Model for Alopecia Areata, J Invest Dermatol , 1991, 96 (5): p. 978; Michie, HJ, et al. , Immunobiological Studies on the Alopecic (DUTY) Rat, Br J Dermatol , 1990, 123 (5): p. 557-67.

6. Examples 6.1. The Conditioning of the Environment

Human dermal fibroblasts were seeded in the substrate of the apparatus described in the '766 patent and which has been described in detail above in Parts 5.3, 5.4 and 5.6. The substrate is inside the box designed to facilitate the three-dimensional tissue growth on its surface, and the cells were cultured in a closed system in DMEM (DMEM = Dulbecco Modified Eagle's Medium Rich in Glucose (BCS (BCS = 10% bovine calf serum supplemented with 2 mM L-glutamine and 50 mg / ml ascorbic acid) a 37 ° C in a humidified atmosphere with 5% CO2. After of 10 days the cell culture was removed, and medium was added without use. The cells were cultured for another 4 days as It has been described above. The resulting conditioned medium, that had been exposed to cell and tissue culture for space of four days (days 10-14), it was then removed from the individual chambers and gathered. The middle conditioning (approximately 5 to 10 liters / tank) was distributed in 20 ml aliquots and was concentrated additionally increasing the concentration from 10 to 20 times using a positive pressure concentration device that had a 10,000 MW cut filter (Amicon, Beverly, MA). The resulting 10 to 20 ml of concentrated conditioning medium were distributed in 1 ml aliquots and frozen at -20ºC for Your analysis A concentration of 1X of conditioned medium results 10X of conditioned medium added to medium base as a solution to 10% (vol / vol). Similarly, a 1X concentration of "medium" or "serum-free medium" results from 10X of medium (ie, base medium) or 10X serum free medium (base medium without serum) added to the base medium as a 10% solution (vol / vol), the which are then used as controls.

6.2. The Proliferative Activity of the Conditioning Medium Three-dimensional 6.2.1. Exposure of fibroblasts and keratinocytes to Conditioned medium

The air conditioner in Part 6.1 was examined to determine its ability to promote the proliferation of human fibroblasts and keratinocytes. Human fibroblasts or human basal keratinocytes were seeded in 96 cavity plates (~ 5,000 cells / cavity) and cultured in glucose-rich DMEM (10% BCS supplemented with 2 mM L-glutamine and 1X antibiotic / antifungal) supplemented with a final concentration of 1X of medium free of serum, medium or three-dimensional conditioned medium as has been described above in Part 6.1. The crops were maintained at 37 ° C in a humidified atmosphere with 5% of CO_ {2} for 3 days.

6.2.2. Cell Proliferation

Cell proliferation was measured using a fluorescence based dye assay and available commercially that measures the total nucleic acid content as estimation of cell proliferation (CyQuant Cell Proliferation Assay Kit, Molecular Probes, Eugene, Or). All the trials were carried out following the manufacturer's instructions. He medium was removed by transfer and the cells were lysed using lysis buffer containing the fluorescent dye green, which was the CyQuant GR dye. The dye has a strong increase in fluorescence when set to cell nucleic acids, and the amount of fluorescence is proportional to the amount of nucleic acid that is present in the sample. The samples were incubated for 5 minutes in absence of light, and the fluorescence of the samples was determined using a microtiter plate reader with appropriate filters for excitation maximums of 480 480 nm and emission of sim 520 nm The amount of nucleic acid in each sample was calculated comparing the amount of fluorescence observed in each cavity with a standard curve obtained using known concentrations of calf thymus DNA as a standard.

As shown in Figure 3, the cells grown in the medium containing the conditioned medium resulted in an increased cell proliferation of both Keratinocyte-like fibroblasts compared to both controls

6.3. Modulation of Collagen Deposition in Tissues By means of three-dimensional conditioning 6.3.1. Wound Healing Applications

The effect of three-dimensional conditioning in the preparation and composition of three-dimensional tissues it was examined by measuring the amount of collagen secreted into the extracellular matrix of tissues grown in the presence of medium free of serum, medium or three-dimensional conditioned medium.

Nylon scaffolds were laser cut in 11 mm x 11 mm squares, washed in 0.5M acetic acid, rinsed extensively in FBS (FBS = fetal bovine serum) and seeded with 12F clinical passage 12F fibroblasts (\ sim 38,000 / cm2). The cultures were carried out in 1 ml of DMEM (10% BCS supplemented with 2 mM L-glutamine and 1x of antibiotic / antifungal) supplemented with a concentration 1X end of serum free medium, medium or conditioned medium three-dimensional as described previously in Part 6.1, with the addition of 50 mg / ml ascorbate in each diet. It was added copper sulfate to a final concentration of 2.5 ng / ml, and a high oxygen content was maintained (of 40%, which is approximately double the atmospheric) by gassing regulated of a standard incubator. The cultures (n = 3 or more) were maintained at 37 ° C in a humidified atmosphere with 5% of CO 2 for 10 days. A control was also included No ascorbate

6.3.2. Collagen Isolation

The collagen was isolated and purified until almost homogeneity from three-dimensional tissue cultures grown in the presence of base medium supplemented with a 1X final concentration of serum-free medium, medium or medium Three-dimensional conditioning as described above. The Purity of the final sample preparations was estimated subjecting the samples of purified collagen to electrophoresis on gradient polyacrylamide-SDS gels, visualizing the different protein bands using blue Coomassie, and estimating the amount of alpha, beta and gamma bands specific for collagen compared to total protein (more ahead). The purification methods produced similar distributions in all samples.

The samples were rinsed in PBS (PBS = phosphate buffered saline) and then in sterile water, and then continuation for 2-6 hours in acid 0.5 M acetic acid. The samples were then digested. overnight in 1 mg / ml pepsin (Worthington, Inc.) in HCl 0.012N at 4 ° C. The samples were clarified by centrifugation at 13,000 rpm at 4 ° C. The collagen was precipitated by 30-60 minutes at 4 ° C after the addition of 5 M NaCl to a final concentration of 0.7 M. The collagen that precipitated was separated by centrifugation at 13,000 rpm at 4 ° C by space of 30 to 60 minutes, and was put back on hold in 0.012 N HCl.

6.3.3. Analysis

Total protein was determined using a set of materials and utensils of colorimetric analysis that is commercially available (Pierce, Inc. BCA assay kit), and analyzes were performed following the instructions of the maker. Bovine skin collagens were used as a pattern (InVitrogen, Carlsbad, CA; Cohesion Technologies, Inc., Palo Alto, CA) to quantify total protein.

The samples (10 mg) were then submitted to analysis by SDS-PAGE with electrophoresis on 3-8% gradient gels. The samples of Isolated collagens were then heated to 95 ° C in buffer of reducing sample. The gels were colored with Blue from Coomassie, discolored and scanned by computer for your display.

As shown in Figure 4, a statistically significant increase (p <0.05) of the Collagen deposition of approximately 50% for crops three-dimensional treated with three-dimensional conditioned medium in Comparison with each control. The activity could not be attributable to the presence of medium or serum alone.

Such increased collagen deposition in vivo has a number of applications, including those for wound healing and wrinkle treatment and contour marks that appear with advanced age, as well as being able to promote deposition. of matrix on the bony prominences that are susceptible to developing bedsores in paralyzed or bedridden patients.

7. Antioxidant activity 7.1. Exposure of Epidermal Skin Cells to the Environment Conditioned

Antioxidant agents have been shown to have the potential to reverse / prevent aging and cellular malignancy. The antioxidant activity of the three-dimensional conditioned medium was measured in epidermal skin cells. Human basal keratinocytes (~ 100,000 / cavity) were cultured in Petri dishes in the presence of MCDB153 medium (KGM, Clonetics, Inc.) supplemented with 1X of three-dimensional conditioned medium and serum-free medium or medium as controls for space of 3 days at 37 ° C in a humidified atmosphere with 5% CO2. The medium was removed from each sample and the cells were isolated from the capsule by incubation in the presence of trypsin (Sanbrook et al ., 1989, Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Lab Press, Cold Spring Harbor, NY), then centrifugation.

7.2. FACS analysis

The isolated cells were incubated in presence of dihydrorrodamine-1,2,3 (Molecular Probes, Eugene, Or) at 37 ° C for 30 minutes and then analyzed by FACS analysis (cell classification activated by fluorescence) using a FACSCAN device Becton-Dickinson (from Franklin Lakes, New Jersey) following the manufacturer's instructions. The amount of oxidation Intracellular of the reduced dihydrorrodamine dye is directly proportional to the amount of fluorescence intracellular detectable of the oxidized state of the dye.

7.3. Results

Human keratinocytes exposed to the environment Cellular conditioning of the Applicants presented a reduction statistically significant (p <0.0003) of 50% of the intracellular oxidation compared to the same cells incubated in the presence of serum-free medium or medium with serum content (see Fig. 5). The difference observed not seems to be attributable to serum factors or ascorbic acid which are present in the control samples. Therefore, topical administration of crop conditioned medium Three-dimensional can be useful as a cosmeceutical for treat or reverse the effects of cells in the process of aging.

8. Test of occlusive patches for the evaluation of the in vivo effects of a biomanipulated cosmeceutical nutrient solution on human skin 8.1. Experimental design

Six adult females were recruited (from 30-60 years) who were in good health and They consisted of undergoing the experiment. The exclusion criteria they included protein sensitivity, diseases of the skin, the presence of skin damage at the test sites or near them, diabetes, kidney, heart disorders or immunological, the use of anti-inflammatory drugs, immunosuppressive, antihistamines or topical or cosmetic and the pregnancy. Test articles were assigned to sites of assay (2 sites, 3.8 cm 2) on the right forearm or left of each subject according to a rotating program to minimize positional or ordinal predisposition. Site 1 went administered vehicle control, and site 2 was administered treatment composition (i.e. conditioned medium). The occlusion patches were made of a cotton buffer not Webril fabric with 0.2 ml of vehicle or composition of treatment. The patches were covered and fastened by a 3M hypoallergenic plastic occlusive tape. The patches of occlusion were positioned daily on the forearms of 3 subjects for 5 consecutive 24-hour periods. To the 3 other subjects were patched daily by space of 12 consecutive 24-hour periods (while still a course of treatment). The day after the last application of The patches are taken from each site a 2 mm biopsy. This protocol was approved by the IRB (IRB = Review Committee Institutional) of the research organization, which was the California Skin Research Institute (of San Diego, Ca.), and fits to Title 21 of the CFR (CFR = Code of Federal Regulations), Parts 50 and 56.

8.2. Evaluations

The observations were roughly classified as brightening, peeling, crushing, cracking, hyperpigmentation and hypopigmentation. Irritation was Visually scored using a 5-point scale and was Numerically classified as erythema, edema, papules and vesicles (> 25% of the patch site) and reactions identifiable (<25% of the patch site), that is, a reaction under which bullae occur with or without oozing, extension and induration. Histological assessment with hematoxylin and eosin performed by a pathologist certified by the committee included parameters for viable epidermal thickness, the epidermal hyperplasia (acanthosis), the thickness of the cell layer granular, inflammatory infiltrate, mitotic figures, the appearance of collagen and elastic fibers and vasculature.

8.3. Results

No adverse events were induced by the medium conditioning or by control in the 3 subjects who received a 5 day treatment The average scores of Daily irritation for nutrient solution (0.3) and controls (0.2) indicated that both sites frequently did not present visible reaction or erythema or had a slight erythema confluent or distributed in zones. Histology (coloration of the collagen with trichrome) revealed a healthy tissue for all The measured parameters. Therefore, the conditioned medium It has pleasant effects on human skin.

9. Modulation of cell behavior human endothelial

The effects of the environment were determined conditioning and human matrix produced by fibroblasts fixed to the matrix in angiogenesis and motility of endothelial cells The conditioned medium was produced by passage through the three-dimensional fibroblast culture here describes (described in Parts 5.3, 5.4 and 5.6) and was concentrated (10X) or lyophilized. The human extracellular matrix was physically removed from the tissue after production.

9.1. Cellular Tubule Formation Assay endothelial

It was used to assess angiogenesis the trial of formation of endothelial cell tubules with cells Endothelial human umbilical vein (HUVEC). The co-culture of HUVEC with lyophilized conditioning caused an increase in tubule formation when compared to the negative control (medium preconditioned) with 4.9 ± 9.31 mm and 0.00 ± 0.00 mm respectively; the concentrated medium increased the formation of tubules up to 44.10 ± 1.75 mm; and the human extracellular matrix increased tubule formation to 39.3 ± 5.6 mm.

9.2. Healing Trial

A confluent layer of cells was scratched endothelial, and the closing speed of the "wound" The result was measured to assess cell motility. The measurement in the "wound" trial consisted of the determination of the closing speed in mm / h (millimeters / hour). Endothelial cells treated with lyophilized medium presented a speed of 25.59 ± 12.907 mm / h, and in the case of the medium concentrated a speed of 39.56 ± 15.87 mm / h was given. Matrix extracellular human did not show an increase in speed in comparison with the negative control (preconditioned medium), with 0.00 mm / h and 27.96 ± 10.01 mm / h for the human extracellular matrix and the negative control, respectively.

Therefore, as the variations illustrate of angiogenesis (tubule formation assay) and of the cellular motility (assessed using the "wound" test), The conditioned medium of the invention is capable of modulating the Human endothelial cell behavior.

Claims (16)

1. A method of preparing a composition Pharmaceutical comprising: (a) culturing three-dimensional fibroblast cells to form a three-dimensional stroma tissue in a cell culture medium sufficient to meet the nutritional needs required for in vitro cell growth and where the stromal cells are attached to a structure composed of a non-living biocompatible material formed in a three-dimensional structure and subsequently surrounding said structure; (b) culturing the cells in vitro until the cell culture medium contains a desired level of extracellular products so that a conditioned medium is formed; (c) separate the conditioned medium from the cells used to condition the medium; Y (d) combine the air conditioner with a Pharmaceutical support 2. The method of claim 1 wherein the conditioned medium is recovered from a flow system continuous. 3. The method of claims 1 or 2 in where the conditioned medium is grown in an environment aseptic. 4. The method according to any of the previous claims wherein the conditioned cellular medium is in the form of or processed in the state of a liquid, solid, lyophilisate, powder, gel or film. 5. The method of any of the above claims wherein the conditioned medium is processed from additional way to concentrate or reduce one or more products content in the middle. 6. The method of claim 5 wherein the method comprises the reduction or enrichment of one or more extracellular products derived from media conditioned by protein separation techniques including chromatography of immunoaffinity, gel chromatography, ion exchange, chromatography of metal chelate affinity, HPLC purification and hydrophobic interaction chromatography. 7. The method of claim 5 wherein the extracellular product (s) is a matrix protein extracellular 8. The method of claim 5 wherein the extracellular product (s) is / are a growth factor, an anti-inflammatory mediator, an enzyme, a cytokine, a hormone, an antigen, an antibody, a factor of coagulation, a regulatory factor, an angiogenic factor or a factor anti-angiogenic 9. The method of any of the above claims where the three dimensional structure It comprises a mesh, a sponge or a polymerized hydrogel. 10. The method of any of the above claims wherein fibroblast cells comprise Genetic engineering cells 11. The method of any of the above claims where genetic engineering cells are transfected with an exogenous gene under the control of an element of expression so that the exogenous gene product is expressed and segregated in the conditioned environment. 12. The method of any of the above claims where parenchymal cells are cultured  in a three-dimensional stroma tissue to form a culture of tissue-specific three-dimensional tissue. 13. The method of any of the above claims wherein parenchyma cells are cells of skin, liver, kidney, neuronal, pancreatic, intestinal, genitourinary, kidney, spleen, bone, marrow Bone or mucosa. 14. The method of any of the above claims wherein parenchyma cells comprise Genetic engineering cells 15. The method of claim 14 wherein genetically engineered cells are transfected with a gene exogenous under the control of an expression element so that the Exogenous gene product is expressed and secreted in the medium conditioned. 16. A method for the isolation of collagen, which includes: (a) precipitation of the pre-collagen of a medium conditioned by a three-dimensional crop at conditions of high salt under neutral pH conditions; (b) separate the propeptides under conditions acidic so that the triple helix of the collagen remains intact; Y (c) precipitate the collagen at high salt concentrations